Articles comprising large-surface-area bio-compatible materials and methods for making and using them

ABSTRACT

The present invention provides articles of manufacture comprising biocompatible nanostructures comprising significantly increased surface area for, e.g., organ, tissue and/or cell growth, e.g., for bone, tooth, kidney or liver growth, and uses thereof, e.g., for in vitro testing of drugs, chemicals or toxins, or as in vivo implants, including their use in making and using artificial tissues and organs, and related, diagnostic, screening, research and development and therapeutic uses, e.g., as drug delivery devices. The present invention provides biocompatible nanostructures with significantly increased surface area, such as with nanotube and nanopore array on the surface of metallic, ceramic, or polymer materials for enhanced cell and bone growth, for in vitro and in vivo testing, cleansing reaction, implants and therapeutics. The present invention provides optically transparent or translucent cell-culturing substrates. The present invention provides biocompatible and cell-growth-enhancing culture substrates comprising elastically compliant protruding nanostructure substrates coated with Ti, TiO 2  or related metal and metal oxide films.

CROSS-REFERENCE TO RELATED APPLICATIONS

This United States utility patent application a continuation of U.S.patent application Ser. No. 12/305,887, filed Sep. 8, 2010, now U.S.Pat. No. 9,149,564, issued Jun. 10, 2015, which is a section 371national phase of PCT international patent application no.PCT/US2007/071947, having an international filing date of Jun. 22, 2007,which claims benefit of priority to U.S. Provisional Patent ApplicationSer. No. 61/816,221, filed Jun. 23, 2006. The aforementionedapplications are expressly incorporated herein by reference in theirentirety and for all purposes.

FIELD OF THE INVENTION

The present invention provides articles of manufacture comprisingbiocompatible nanostructures comprising significantly increased surfacearea for, e.g., organ, tissue and/or cell growth, e.g., for bone, tooth,kidney or liver growth, and uses thereof, e.g., for in vitro testing ofdrugs, chemicals or toxins, or as in vivo implants, including their usein making and using artificial tissues and organs, and relateddiagnostic, screening, research and development and therapeutic uses,e.g., as drug delivery devices. The present invention providesbiocompatible nanostructures with significantly increased surface area,such as with nanotube and nanopore array on the surface of metallic,ceramic, or polymer materials for enhanced cell and bone growth, for invitro and in vivo testing, cleansing reaction, implants andtherapeutics. The present invention provides optically transparent ortranslucent cell-culturing substrates. The present invention providesbiocompatible and cell-growth-enhancing culture substrates comprisingelastically compliant protruding nanostructure substrates coated withTi, TiO₂ or related metal and metal oxide films.

BACKGROUND OF THE INVENTION

It is known that the nano-scaled materials exhibit extraordinaryelectrical, optical, magnetic, chemical and biological properties, whichcannot be achieved by micro-scaled or bulk counterparts. The developmentof nano-scaled materials has been intensively pursued in order toutilize such properties for various technical applications includingbiomedical and nano-bio applications.

Two-dimensional and three-dimensionally cultured cells are useful notonly for liver cell related applications, but for producing a number ofother cells in a healthy and accelerated manner. There are needs tosupply or implant various types of cells including bone cells, livercells, kidney cells, blood vessel cells, skin cells, periodontal cells,stem cells, and other human or animal organ cells.

There is a critical need for an artificial liver device that can removetoxins and improve immediate and long-term survival of patientssuffering from liver disease. An artificial liver device can be usefulas a temporary artificial liver for patients awaiting a livertransplant, and also provide support for post-transplantation patientsuntil the grafted liver functions adequately to sustain the patient. Oneof the major roadblocks to the development of an effective artificialliver device is the lack of a satisfactory liver cell line that canprovide the functions of a liver.

A fast growth and supply of cells especially rare cells, such as stemcell enrichment, can be crucial for many potential therapeuticapplications as well as for enhancing the speed of advances in stem cellscience and technology. In addition, fast detection and diagnosis ofdisease cells or possible bio-terror cells (such as epidemic diseases,anthrax or SARS) from a very small or trace quantity of available cellscan be accomplished if the cell growth speed can be accelerated.

SUMMARY

The invention provides new biomaterials structures havingmacro-micro-nano combined features strongly bonded onto and protrudingabove the surface of the structure, e.g., an implant or cell-growthsubstrate surface of the invention. The invention also provides analternative embodiment of large-surface-area, free-standingconfiguration of loose crumbled wire mesh, short fiber or loose powderconfigurations instead of having them bonded/attached onto a solidimplant or substrates.

The invention provides compositions comprising large-surface-area, thin,macro or microscale members comprising hairy, gauge, wire, woven wire,spring, ribbon or particulate array configurations of Ti or otherrelated metals, alloys and/or ceramics that are directly and stronglybonded onto a Ti-base or alloy-base implants or cell-growth substrates;and in one aspect, with the surface of each of the members having a highdensity array of titanium oxide nanotube or titanium oxide coverednanopore surface.

In alternative aspects, the thickness (or “thinness”, depending on thecontext) of a coating on the surface of any product of manufacture ofthis invention (including implants, devices, etc.) can be in the rangefrom about 1 to 100 nm, 1 to 50 nm, or about 1 to 20 nm, or inalternative embodiments: at least 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46,47, 48, 49 or 50 or more nm. The thickness or “thinness” (bothreferencing the amount (or vertical dimension) of coating on a surface)can be even, or not even, over any particular portion, or all of asurface of a product of manufacture of this invention. The coating canbe multi-laminar (multilayered) with one or a mixture of compounds, asdescribed herein.

An alternative embodiment comprises loose, short-fibers or particles ofTi with a large-surface-area, high density array of titanium oxidenanotubes or titanium oxide covered nanopores.

Biomaterials of the invention can exhibit enhanced cell adhesion andaccelerated cell growth characteristics as well as enhanced bone growth.In one embodiment, the thin Ti members and or in another aspect, thebase Ti-base implants, can have a cell-adhesion-improving andcell-growth-accelerating surface nanostructure of TiO₂ nanotubes orTiO₂-covered nanopores with a density of at least about 0.25×10⁸/cm², orin another aspect, at least about 1×10⁸/cm², or at least between about0.2×10⁸/cm² to 2×10⁸/cm².

In alternative aspects, the invention provides micro-nano or macro-nanocombined biomaterials implants with protruding structural features abovethe implant surface; and in alternative embodiments these can provideadvantageous characteristics for bio applications such as furtherimproved stability, biocompatibility and mechanical lock-in reliabilityat the implant-cultured bone/cell interface, as well as substantiallyaccelerated cell/bone growth accelerating characteristics due to theTiO₂ nanotube and related structures.

Exemplary biomaterials used to practice this invention include: (i) asurface structure with fast-growing cells or bones encircling orsurrounding micro or macro members; in one aspect this providessignificantly enhanced mechanical lock-in structure for improvedmechanical strength on tensile or shear strain; (ii) bonded Ti membershaving compliant or springy structures which can accommodatestrains/stresses during the early stage of bone growth to reduce a riskof catastrophic bone-implant interface failure; (iii) structurescomprising micro/macro Ti members such as mesh screens, ribbons or wirearrays strongly bonded serving as on Ti implant surface, which inalternative aspects can serve as efficient, high-density structuralreinforcement within the grown bone, (iv) loose configured Tishort-fibers, fragments, particles with high-density surfacenanostructure of TiO₂ nanotubes or TiO₂-covered nanopores, which inalternative aspects can serve as efficient and convenient additives tobone-growth-accelerating composites or cements, for repair of orthopedicor dental bones; (v) micro-nano or macro-nano combined structures, whichin alternative embodiments can comprise growth factors and/or otherbiological agents such as antibiotics, genes, proteins, drugs, magneticnanoparticles added inside the nanopores or nanotubes for, e.g., furtheraccelerated cell growth, healthy cell growth, drug release for varioustherapeutic uses; (vi) the added micro or macro Ti members with largesurface area, which in alternative aspects allows easier growth ofthree-dimensional cell, organ or bone structures; (vii) biocompatibleTiO₂-nanotube type, TiO₂-nanofiber type or TiO₂-nanopore type surfaces,which in alternative aspects can also be applied onto the surface ofother non-Ti-based implants of metallic, ceramic, semiconductor, orpolymer materials by thick film coating followed by anodization andoptional crystallization heat treatment.

These structures of the invention can be useful for rapid production ofhealthy cells including liver cells, bone cells, kidney cells, bloodvessel cells, skin cells, periodontal cells, stem cells and other rarecells, as well as rapid formation/growth of strongly adherent bones. Inone aspect, structures the invention can be useful for reliable andfaster orthopedic or dental bone repair, for preparation of partial orfull implant organs for in vivo insertion, or ex vivo operation as anartificial liver or kidney, for externally controllable drug release andtherapeutic treatments, for efficient toxicity testing of drugs andchemicals, and for diagnosis/detection of disease or forensic cells.

The invention also provides various methods of manufacture, methods ofcell culturing, method of implant applications using the inventive,cell/bone-growth accelerating biomaterials of this invention.

The invention also provides products of manufacture, e.g., as drugdelivery devices, comprising a biocompatible surface comprising (a) aleast a portion of its surface area comprising (i) thin, macro ormicroscale members comprising hairy, gauge, wire, woven wire, spring,ribbon, powder or particulate array configurations, or comprising a Tior other related metal material, an alloy and/or a ceramic; (ii) loose,short-fibers or particles of Ti; (iii) micro-wires or micro-ribbons;(iv) spring-like fiber or mesh screen shapes comprising a Ti or otherrelated metal material, an alloy and/or a ceramic; or (v) structures asillustrated in FIGS. 1, 2, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 and/or16; and, (b) (i) a biocompatible vertically aligned nanotube arraystructure on a biocompatible substrate comprising a laterally separatednanotube arrangement; (ii) a lock-in nanostructure comprising aplurality of nanopores or nanotubes, wherein the nanopore or nanotubeentrance has a smaller diameter or size than the rest (the interior) ofthe nanopore or nanotube to exhibit a re-entrant configuration; (iii) adual structured biomaterial comprising (A) micro- or macro-pores,wherein the micro or macro pores has an average diameter, or equivalentdiameter if the pores are not circular, in the range of between about0.5-1,000 μm, or between about 1-100 μm, and optionally the entrances ofthe micro or macro pores have a smaller diameter or size than the rest(the interior) of the micro or macro pores; and, (B) a surface areacovered with nanotubes, optionally TiO₂ nanotubes, having an averagepore diameter in the range of between about 30-600 nm; (iv) abiomaterial having a surface comprising a plurality of enlarged diameternanopores and/or nanotubes, wherein the nanopores and/or nanotubescomprise at least 150 nm, or optionally at least 200 nm, or at least 400nm; (v) an array comprising a solid substrate and a plurality ofvertically aligned, laterally spaced, nanotubes associated with thesubstrate, wherein each nanotube comprises a nanopore; (vii) an arraycomprising a solid substrate and a plurality of vertically aligned,laterally spaced, nanotubes associated with the substrate, wherein eachnanotube comprises a nanopore and wherein the array comprises a cell,wherein optionally the cell is suitable for implantation, and optionallythe cell is suitable for implantation and regeneration of an organ or adental tissue in a subject; (viii) an array comprising a solid substrateand a plurality of vertically aligned, laterally spaced, nanotubesassociated with the substrate, wherein each nanotube comprises ananopore and wherein the array comprises a cells suitable forimplantation and regeneration of a bone and/or a joint tissue in asubject; (ix) an array comprising a solid substrate and a plurality ofvertically aligned, laterally spaced, nanotubes associated with thesubstrate, wherein each nanotube comprises a nanopore and wherein thearray comprises one or more biologically active agents selected from thegroup consisting of therapeutic drugs, growth factors, proteins,enzymes, hormones, nucleic acids, RNA, DNA, genes, vectors, antibioticsor antibodies, small molecules, radioisotopes and magneticnanoparticles; (x) a two or a three-dimensional array comprising (A) asolid substrate comprising Ti wires, ribbons or rods, or any combinationthereof; and (B) a plurality of vertically aligned, laterally spaced,nanotubes associated with the substrate, wherein each nanotube comprisesa nanopore; (xi) a product of manufacture comprising a size-randomizedand shape-randomized nanopore- or nanotube-comprising surface made by amethod comprising the following steps (A) providing a compositioncomprising a Ti or Ti oxide surface, (B) depositing a semi-wettablecoating on the Ti or Ti oxide surface by employing lithographicpatterning or by a thin film deposition technique, wherein the coatingdecomposes into nano- or micro-islands of local etch masks, and (C)chemical etching or electrochemical anodization of the etch-maskedsurface, thereby generating a size-randomized and shape-randomizednanopore- or nanotube-comprising surface; or (xii) a combinationthereof. In one aspect of the product of manufacture, the thin, macro ormicroscale members and/or the nanotube array structure comprise Ti andTi oxide, Zr, Hf, Nb, Ta, Mo, W and/or their alloys or oxides of thesemetals, and/or alloys; and optionally having a thickness of at least 5nm; and optionally having a coating coverage of at least 80% of thenanotube or nanopore surfaces, wherein the matrix material comprises Ti,Zr, Hf, Nb, Ta, Mo, W, and/or their oxides, or alloys of these metalsand oxides, and/or Si, Si oxide, Al, Al oxide, carbon, diamond, noblemetals, Au, Ag, Pt and/or their alloys, polymer or plastic materials, orcomposite metals, ceramics and/or polymers.

Alternative embodiments of the products of manufacture of the inventionare optically transparent or translucent cell-culturing substrates withnano imprint patterned nanostructures. In this aspect, opticaltransparency of a cell culture substrate is an important characteristic,as it allows a microscopic examination of the cell behavior usinginverted microscope with transmitted light illumination. The surface ofsuch a nanostructure is coated with an optically transparent ortranslucent, very thin film of Ti or Ti-base alloys (e.g., Ti—Al—Valloys), other refractory metals (e.g., Zr, Nb, Hf, Ta, W and theiralloys), or TiO₂, NbZO₅, ZrO₂, HfO₂, Ta₂O₅, W₂O₃ or mixed alloy oxide.In alternative aspects, the thickness of the Ti or TiO₂ related coatingis between about 1 to 50 nm, or between about 1 to 20 nm. The relatedcoating can comprise (i) a Ti, Zr, Hf, Nb, Ta, Mo and/or W metal; (ii)an oxide of (i); (iii) an alloy of (i); (iv) a Si, a Si oxide, an Al, anAl oxide, a carbon, a diamond, a noble metal, an Au, an Ag, a Pt and/oran Al, Au, an Ag, a Pt alloy, a polymer or a plastic material, acomposite metal, a ceramic, a polymer and/or a combination thereof.

Alternative embodiments of the products of manufacture of the inventioncomprise an elastically compliant nanostructure substrate coated withTi, TiO₂ or related metal and metal oxide or nitride films, which cancomprise (i) a Ti, Zr, Hf, Nb, Ta, Mo and/or W metal; (ii) an oxide of(i); (iii) an alloy of (i); (iv) a Si, a Si oxide, an Al, an Al oxide, acarbon, a diamond, a noble metal, an Au, an Ag, a Pt and/or an Al, Au,an Ag, a Pt alloy, a polymer or a plastic material, a composite metal, aceramic, a polymer and/or a combination thereof. As the stress or strainthat the growing/propagating cells experience has a tremendous effect onthe cell growth behavior. By providing elastically soft substrate whichis made even more flexible by virtue of added surface nanostructure, afurther enhanced cell growth is obtained.

The invention provides products of manufacture comprising abiocompatible surface, wherein at least a portion of, or all of, thesurface area of the biocompatible surface comprises or is covered orcoated by structures comprising:

(A)(i) a plurality of thin, macro or microscale members in a hairy, agauge, a wire, a woven wire, a spring, a ribbon, a powder, a flakeshaped structure and/or a particulate array configuration, wherein thethin, a macro or a microscale member comprises a Ti, Zr, Hf, Nb, Ta, Moand/or W metal material, a Ti, Zr, Hf, Nb, Ta, Mo and/or W alloy, a Ti,Zr, Hf, Nb, Ta, Mo and/or W oxide or nitride, and/or stainless steel orceramic, wherein the plurality of thin, macro or microscale members arefixed or loosely placed, or a combination thereof, on the biocompatiblesurface;

(ii) a plurality of loose, short-fibers, or flake shaped structures, orparticles of Ti, Zr, Hf, Nb, Ta, Mo and/or W metal material, a Ti, Zr,Hf, Nb, Ta, Mo and/or W alloy, a Ti, Zr, Hf, Nb, Ta, Mo and/or W oxideor nitride, and/or stainless steel or ceramic, wherein optionally thefibers or particles are straight, curved and/or bent, and optionally thespring-like fibers or mesh screen shapes are fixed or loosely placed, ora combination thereof, on the biocompatible surface;

(iii) a plurality of micro-wires, micro-fibers or micro-ribbons, whereinoptionally the wires, ribbons or fibers are straight, curved and/orbent, wherein optionally the micro-wires, micro-fibers or micro-ribbonsare fixed or loosely placed, or a combination thereof, on thebiocompatible surface;

(iv) a plurality of spring-like fibers or mesh screen shapes comprisinga Ti or metal material, an alloy and/or a ceramic, wherein optionallythe spring-like fibers or mesh screen shapes are fixed or looselyplaced, or a combination thereof, on the biocompatible surface; (v) astructure as illustrated in FIGS. 1, 2, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15 and/or 16;

(vi) any of the structures of (i) to (v) in the form of ahigh-surface-area wire array, a mesh screen array, a particle assemblyarray or a combination thereof;

(vii) any combination of the structures of (i) to (vi), and optionallywherein any combination of the structures of (i) to (vi) are fixed orloosely placed as a loose deposit, a loose powder, a loose film, a looseparticle, a loose short-fiber or a loose flake, or any combinationthereof, on the biocompatible surface;

and the biocompatible surface comprises structures comprising:

(B) (i) a biocompatible vertically aligned nanotube array structure on abiocompatible substrate comprising a laterally separated nanotubearrangement;

(ii) a lock-in nanostructure comprising a plurality of nanopores ornanotubes, wherein the nanopore or nanotube entrance has a smallerdiameter or size than the rest (the interior) of the nanopore ornanotube to exhibit a re-entrant configuration;

(iii) a dual structured biomaterial comprising (A) micro- ormacro-pores, wherein the micro or macro pores has an average diameter,or equivalent diameter if the pores are not circular, in the range ofbetween about 0.5 to 1,000 μm, or between about 1 to 100 μm, andoptionally the entrances of the micro or macro pores have a smallerdiameter or size than the rest (the interior) of the micro or macropores; and, (B) a surface area covered with nanotubes, optionally TiO₂nanotubes, having an average pore diameter in the range of between about30 to 600 nm;

(iv) a plurality of enlarged diameter nanopores and/or nanotubes,wherein the nanopores and/or nanotubes comprise at least 150 nm, oroptionally at least 200 nm, or at least 400 nm;

(v) an array comprising a solid substrate and a plurality of verticallyaligned, laterally spaced, nanotubes associated with the substrate,wherein each nanotube comprises a nanopore;

(vii) an array comprising a solid substrate and a plurality ofvertically aligned, laterally spaced, nanotubes associated with, orbuilt into or onto, the substrate, wherein each nanotube comprises ananopore, and the array comprises a cell, wherein optionally the cell issuitable for implantation, and optionally the cell is suitable forimplantation and regeneration of an organ or a dental tissue in asubject;

(viii) an array comprising a solid substrate and a plurality ofvertically aligned, laterally spaced, nanotubes associated with thesubstrate, wherein each nanotube comprises a nanopore and wherein thearray comprises a cell suitable for implantation and regeneration of abone and/or a joint tissue in a subject;

(ix) an array comprising a solid substrate and a plurality of verticallyaligned, laterally spaced, nanotubes associated with the substrate,wherein each nanotube comprises a nanopore and wherein the arraycomprises one or more biologically active agents selected from the groupconsisting of a therapeutic drug, a growth factor, a protein, an enzyme,a hormone, a nucleic acid, an RNA, a DNA, a gene, a vector, a phage, anantibiotic, an antibody, a small molecule, a radioisotope and a magneticnanoparticle or particle;

(x) a two or a three-dimensional array comprising (A) a solid substratecomprising Ti, Zr, Hf, Nb, Ta, Mo and/or W wires, ribbons or rods, orany combination thereof; and (B) a plurality of vertically aligned,laterally spaced, nanotubes associated with the substrate, wherein eachnanotube comprises a plurality of nanopores;

(xi) a product of manufacture comprising a size-randomized andshape-randomized nanopore- or nanotube-comprising surface made by amethod comprising the following steps (A) providing a compositioncomprising a Ti, Zr, Hf, Nb, Ta, Mo and/or W metal surface; a Ti, Zr,Hf, Nb, Ta, Mo and/or W alloy surface; and/or, a Ti, Zr, Hf, Nb, Ta, Moand/or W oxide or nitride, and/or stainless steel or ceramic, (B)depositing a semi-wettable coating on a metal, alloy or oxide surface of(A) by employing lithographic patterning or by a thin film depositiontechnique, wherein the coating decomposes into nano- or micro-islands oflocal etch masks, and (C) chemical etching or electrochemicalanodization of the etch-masked surface, thereby generating asize-randomized and shape-randomized nanopore- or nanotube-comprisingsurface;

(xii) an elastically compliant nanostructure substrate comprising anycombination of (i) to (xi), and/or a structure of (A);

(xiii) any combination of (A) or (B)(i) to (xii), and/or a structure of(A), comprising or configured as an optically transparent or translucentcell-culturing substrate, which optionally is a nano imprint patternednanostructure;

(xiv) any combination of (A) or (B)(i) to (xiii), in the form of aparticle aggregate or a mesoporous structure, or comprising a nanowireor ribbon forest, or comprising directionally etched porous materials ora porous thin film;

(xv) any combination of (A) or (B) (i) to (xiv), wherein the structurespartially or completely coating the biocompatible surfaces have athickness of at least about 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 or more nm,or a thickness of between about 1 to 10 nm, or a thickness of betweenabout 1 to 15 nm, or a thickness of between about 1 to 20 nm; or

(xvi) any combination of any of these structural embodiments of theinvention.

In alternative embodiments of the products of manufacture of theinvention, the thin, macro or microscale members and/or the nanotubearray structure, or a structure of the invention, comprises a Ti, Zr,Hf, Nb, Ta, Mo and/or W metal; a Ti, Zr, Hf, Nb, Ta, Mo and/or W alloy;and/or, a Ti, Zr, Hf, Nb, Ta, Mo and/or W oxide or nitride, and/orstainless steel or ceramic. In alternative embodiments, the thin, macroor microscale members and/or the nanotube array structure have athickness of at least about 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 or more nm,or a thickness of between about 1 to 10 nm, or a thickness of betweenabout 1 to 15 nm, or a thickness of between about 1 to 20 nm.

In alternative embodiments, at least 50%, 51%, 52%, 53%, 54%, 55%, 56%,57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%,71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%,85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99%, or more, or all, of the biocompatible surface or the nanotube ornanopore surface comprises or is covered or coated by a structure of theinvention.

In alternative embodiments, the matrix material, or a structure of theinvention, comprises (i) a Ti, Zr, Hf, Nb, Ta, Mo and/or W metal; (ii)an oxide of (i); (iii) an alloy of (i); (iv) a Si, a Si oxide, an Al, anAl oxide, a carbon, a diamond, a noble metal, an Au, an Ag, a Pt and/oran Al, Au, an Ag, a Pt alloy, a polymer or a plastic material, acomposite metal, a ceramic, a polymer and/or a combination thereof.

In alternative embodiments, the products of manufacture of the inventionfurther comprise a bone cell, a liver cell, a kidney cell, a bloodvessel cell, a skin cells, a periodontal cell or a periodontal tissuecell, a stem cell, an organ cell, or wherein the cell is a bone cell, aliver cell, a kidney cell, a blood vessel cell, a skin cells, an organcell; or, further comprising a plurality of cells, wherein the cellscomprise bone cells, liver cells, liver parenchymal cells, endothelialcells, adipocytes, fibroblastic cells, Kupffer cells, kidney cells,blood vessel cells, skin cells, periodontal cells, odontoblasts,dentinoblasts, cementoblasts, enameloblasts, odontogenic ectomesenchymaltissue, osteoblasts, osteoclasts, fibroblasts, and other cells andtissues involved in odontogenesis or bone formation and/or stem cells,and other human or animal organ cells, or the cells are embryonic oradult stem cells, or a combination thereof. The cell can be a human oran animal cell, or the product of manufacture further comprises a humanor an animal cell.

In alternative embodiments, the products of manufacture of the inventionfurther comprise a hydroxyapatite, a bio-degradable polymer, or abio-compatible or bio-inert bone cement; or further comprising abiological agent, wherein optionally the biological agent comprises agrowth factor, a collagen, a nucleic acid, an antibiotic, a hormone, adrug, a magnetic particle, a metallic particle, a ceramic particle, apolymer particle, a drug delivery particle.

The invention provides drug delivery devices comprising a product ofmanufacture of the invention. The invention provides orthopedic implantsor dental implants comprising a product of manufacture of the invention,and optionally the orthopaedic (orthopedic) implant or dental implantcomprises a plurality of cells, and optionally the cells comprise bonecells, liver cells, liver parenchymal cells, endothelial cells,adipocytes, fibroblastic cells, Kupffer cells, kidney cells, bloodvessel cells, skin cells, periodontal cells, odontoblasts,dentinoblasts, cementoblasts, enameloblasts, odontogenic ectomesenchymaltissue, osteoblasts, osteoclasts, fibroblasts, and other cells andtissues involved in odontogenesis or bone formation and/or stem cells,and other human or animal organ cells, or the cells are embryonic oradult stem cells, or a combination thereof.

The invention provides disease detection devices comprising a product ofmanufacture of the invention.

The invention provides artificial tissue or organs comprising a productof manufacture of the invention, and optionally the artificial tissue ororgan comprises a plurality of cells, and optionally the cells comprisebone cells, liver cells, liver parenchymal cells, endothelial cells,adipocytes, fibroblastic cells, Kupffer cells, kidney cells, bloodvessel cells, skin cells, periodontal cells, odontoblasts,dentinoblasts, cementoblasts, enameloblasts, odontogenic ectomesenchymaltissue, osteoblasts, osteoclasts, fibroblasts, and other cells andtissues involved in odontogenesis or bone formation and/or stem cells,and other human or animal organ cells, or the cells are embryonic oradult stem cells, or a combination thereof.

The invention provides bioreactors comprising a product of manufactureof the invention. The invention provides biomimetic arrays, orartificially constructed cell cultures, comprising a product ofmanufacture of the invention, and optionally also comprising at least acell for performing drug or chemical toxicity testing, and optionallythe cell is a liver cell or liver parenchymal cells.

The invention provides methods for evaluating a drug or a chemical, apesticide or herbicide, a toxin, a poison, a pharmaceutical, a cosmetic,a polymer or an injection fluid, comprising applying the drug, chemical,toxin, poison, pharmaceutical, pesticide or herbicide, cosmetic, polymeror injection fluid to a biomimetic array, or artificially constructedcell culture, comprising use of a product of manufacture of thisinvention, wherein optionally method comprises the testing of a new drugor a chemical for safety and/or toxicity issues, and optionally thechemical comprises a toxin, a poison, an allergen, a biological warfareagent, an infectious disease agent or an irritating agent.

The invention provides methods for diagnosing or detecting a diseasecomprising implanting a product of manufacture of the invention in asubject, wherein optionally the implant is a biochip comprising adisease detection compound or device.

The invention provides methods for detecting an infectious diseaseagent, or a biological warfare or a bio-terror agent, the methodcomprising providing a product of manufacture of the invention, whereinthe product of manufacture comprises an infectious disease agent,biological warfare or bio-terror agent detection compound or device, andoptionally the product of manufacture is an implant.

The invention provides systems for growing and harvesting selectedcells, the system comprising: (a) a product of manufacture of theinvention operably associated with a device for removing the cells ortissue from the product of manufacture; and (b) a computer operablyassociated with a), wherein the computer comprises instructions forautomatically contacting the cells with a suitable growth media and forharvesting the mature cells.

The invention provides methods for treating a cell proliferationdisorder, the method comprising: (a) implanting a product of manufactureof the invention, into a subject, wherein the product of manufacturecomprises a biological agent for treating the cell proliferationdisorder, and optionally the product of manufacture is implanted at ornear the site of a cell proliferation disorder; and (b) contacting theproduct of manufacture with magnetic agitation, wherein optionally theagitation accelerates biological agent release; and optionally providesmagnetic hyperthermia treatment at the site of implantation, andoptionally the magnetic agitation comprises external stimulation of themagnetic nanoparticles by alternating current (AC) magnetic field; andoptionally the biological agent is released from the array by mechanicalagitation/movement of the magnetic particles or by heating of thecomposition resulting from the AC magnetic field.

The invention provides methods for selectively releasing a biologicalagent in a subject, the method comprising (a) implanting a product ofmanufacture of the invention, in a subject, wherein the product ofmanufacture comprises a biological agent in a colloidal composition;and, (b) contacting the product of manufacture with ultrasonic ormagnetic agitation of the colloidal composition, wherein the biologicalagent is released from the product of manufacture; and optionally themagnetic nanoparticle is selected from the group consisting ofiron-oxide particles of magnetite (Fe₃O₄) or maghemite (γ-Fe₂O₃), andoptionally the magnetic nanoparticle is about 5 to 50 nm in averagediameter.

The invention provides methods for accelerating the growth of cells, themethod comprising contacting the cells with a product of manufacture ofthe invention, in the presence of a nutrient fluid suitable forsustaining growth of the cells.

The invention provides multi-functional implant devices comprising aproduct of manufacture of the invention, wherein the product ofmanufacture comprises a biologically active agent selected from thegroup consisting of a pharmaceutical composition, a therapeutic drug, acancer drug, a growth factor, a protein, an enzyme, a hormone, a nucleicacid, an antibiotic, an antibody, a nanoparticle and a biologicallyactive material. In one aspect, of the multi-functional implant device,the product of manufacture comprises a colloidal liquid comprising thebiologically active agent, and the product of manufacture is designedfor externally controlled release of the colloidal liquid uponapplication of ultrasonic or magnetic stimulation; and optionally thecolloidal liquid comprises a biologically active agent and magneticnanoparticles; and the magnetic nanoparticles are selected from thegroup consisting of biocompatible iron-oxide particles of magnetite(Fe₃O₄) and maghemite (γ-Fe₂O₃); and optionally the size of the magneticnanoparticles is from about 5 to 50 nm in diameter. In one aspect, ofthe multi-functional implant device, the product of manufacturecomprises nanotubes, and a cap is deposited at the upper end of ananotube by an oblique incident sputter deposition on a stationary or arotating substrate; and optionally the cap is narrowed such that acolloidal liquid is retained in the nanotube before external stimulationfor controlled release.

The invention provides methods of externally controlled release of acolloidal liquid into a subject comprising applying external stimulationby alternating current magnetic field to the multi-functional implantdevice of the invention, wherein the magnetic field causes agitation,movement and heat production from the magnetic nanoparticles comprisedin the colloidal liquid resulting in its release from the implantdevice.

The invention provides methods for ameliorating or treating cancer,wherein the multi-functional implant device of the invention isimplanted into a subject at the site of cancer; and optionally externalstimulation is applied resulting in the local delivery of anti-cancerdrugs and magnetic hyperthermia treatment.

The invention provides methods of cell proliferation comprising aproduct of manufacture of the invention, and adherent cells, whereinupon adhesion the cells are induced to proliferate; and optionally thecells are grown in vivo, ex vivo or in vitro, and optionally afterproliferation, the cells are harvested.

The invention provides analytical diagnostic biochips comprising aproduct of manufacture of the invention, wherein the biochip is used forthe rapid diagnosis or detection of diseased cells, cells involved in aninfectious or an epidemic disease or exposed to a chemical or a toxicagent, or cells exposed to a biological warfare agent, or cells that arerelated to forensic investigations.

The invention provides methods for making a product of manufacture ofthe invention, comprising simple dropping of the structures of theinvention onto the biocompatible surface, and bonding the structuresonto the surface under compression or by utilizing electric arc spotwelding or heating to high temperature for diffusion bonding.

The invention provides nano-patterned, see-through cell culturesubstrates comprising a polymer, polycarbonate, plastic or glass basecoated with is covered or coated by a structure comprising: (i) aplurality of thin, macro or microscale members in a hairy, a gauge, awire, a woven wire, a spring, a ribbon, a powder, a flake shapedstructure and/or a particulate array configuration, wherein the thin, amacro or a microscale member comprises a Ti, Zr, Hf, Nb, Ta, Mo and/or Wmetal material, a Ti, Zr, Hf, Nb, Ta, Mo and/or W alloy, a Ti, Zr, Hf,Nb, Ta, Mo and/or W oxide or nitride, and/or stainless steel or ceramic,wherein the plurality of thin, macro or microscale members are fixed orloosely placed, or a combination thereof, on the biocompatible surface;(ii) a plurality of loose, short-fibers, or flake shaped structures, orparticles of Ti, Zr, Hf, Nb, Ta, Mo and/or W metal material, a Ti, Zr,Hf, Nb, Ta, Mo and/or W alloy, a Ti, Zr, Hf, Nb, Ta, Mo and/or W oxideor nitride, and/or stainless steel or ceramic, wherein optionally thefibers or particles are straight, curved and/or bent, and optionally thespring-like fibers or mesh screen shapes are fixed or loosely placed, ora combination thereof, on the biocompatible surface; (iii) a pluralityof micro-wires, micro-fibers or micro-ribbons, wherein optionally thewires, ribbons or fibers are straight, curved and/or bent, whereinoptionally the micro-wires, micro-fibers or micro-ribbons are fixed orloosely placed, or a combination thereof, on the biocompatible surface;(iv) a plurality of spring-like fibers or mesh screen shapes comprisinga Ti or metal material, an alloy and/or a ceramic, wherein optionallythe spring-like fibers or mesh screen shapes are fixed or looselyplaced, or a combination thereof, on the biocompatible surface; (v) astructure as illustrated in FIGS. 1, 2, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15 and/or 16; (vi) any of the structures of (i) to (v) in the formof a high-surface-area wire array, a mesh screen array, a particleassembly array or a combination thereof; (vii) any combination of thestructures of (i) to (vi), and optionally any combination of thestructures of (i) to (vi) are fixed or loosely placed, or a combinationthereof, on the biocompatible surface.

The invention provides optically transparent or translucentcell-culturing substrates with nano imprint patterned nanostructurecomprising a product of manufacture of the invention. The inventionprovides biocompatible and cell-growth-enhancing culture substratescomprising an elastically compliant protruding nanostructure substratecoated with Ti, TiO₂ or related metal and metal oxide films (e.g.,comprising a Ti, Zr, Hf, Nb, Ta, Mo and/or W metal material, a Ti, Zr,Hf, Nb, Ta, Mo and/or W alloy, a Ti, Zr, Hf, Nb, Ta, Mo and/or W oxideor nitride, and/or stainless steel or ceramic), wherein thenanostructure comprises a product of manufacture of the invention.

The invention provides methods of making a porous biomaterial surfacenanostructure capable of controlled delivery of biological agents usinga product of manufacture of the invention, by additional obliqueincidence sputtering or evaporation, quick electroplating, quickelectroless plating, or quick dipping in adhesives for partially cappingthe entrance of the pores to reduce the pore-entrance-diameter, followedby inserting biological agent into the nanopores. In alternative aspect,the biological agent is selected from a list of a growth factor, acollagen, a nucleic acid, an antibiotic, a hormone, a drug, magneticparticles, metallic particles, ceramic particles, polymer particles anda combination thereof; or the magnetic particles, metallic particles,ceramic particles, or polymer particles are pre-inserted before the porediameter reducing cap material is deposited to minimize inadvertentrelease of the particles outside the biomaterial surface; or thebiomaterial surface nanostructure with nanotube, nanowire, or nanoporeconfiguration is made from a substrate material comprising Ti, Zr, Hf,Nb, Ta, Mo or W metal material, a Ti, Zr, Hf, Nb, Ta, Mo or W alloy, ora combination thereof, by a method comprising one or more of processesselected from DC or RF sputter deposition, oblique incident evaporation,chemical vapor deposition, laser surface melting and solidification, RFsurface melting and solidification, chemical etching,patterned-mask-guided chemical, reactive ion etching and a combinationthereof; or the porous biomaterial is in bulk or thick filmconfiguration and is made of non refractory metal related materialscomprising silicon, polymer, plastic, glass or ceramic material, and thesurface of the biomaterial is pre-coated with a biocompatible,cell-culture-enhancing thin film layer comprising Ti, Zr, Hf, Nb, Ta, Moor W metal material, a Ti, Zr, Hf, Nb, Ta, Mo or W alloy, Ti, Zr, Hf,Nb, Ta, Mo or W oxide, or Ti, Zr, Hf, Nb, Ta, Mo or W nitride, andwherein the pre-coating is made by a method comprising a physicaldeposition method comprising sputtering, evaporation or atomic layerdeposition, or a chemical deposition method selected from CVDdeposition, electrodeposition or electroless deposition; or wherein theporous biomaterial surface is in a configuration of macro-, micro- ornano-particle aggregate and is made by a method comprising sintering orgluing of particles, which optionally can comprise a mesoporous carbon,mesoporous silicon, mesoporous metal, mesoporous ceramic or mesoporouspolymer onto a rigid surface.

The invention provides methods of remote-operating drug delivery systemcomprising an array of magnetic nano-ribbon, magnetic micro-ribbon,magnetic nanowire, magnetic micro-wire, carbon nanotubes coated withmagnetic material by applying remote magnetic field to sequentially movethe magnetic elements.

The invention provides methods of making loose particle, flake, or fiberbased large-surface-area, biocompatible, cell-culture-enhancing orbone-growth-enhancing surface having a nanotube covered structurecomprising: (a) selecting a material comprising Ti, Zr, Hf, Nb, Ta, Moor W metal material; a Ti, Zr, Hf, Nb, Ta, Mo or W alloy; or Ti, Zr, Hf,Nb, Ta, Mo or W oxide, or Ti, Zr, Hf, Nb, Ta, Mo or W nitride; and, (b)introducing a relative rotational, lateral or shaking movement betweenthe particles, flakes or fibers and the electrode so that the surface ofthe Ti and refractory metal loose particles is anodized to formnanotube-covered surface.

The invention provides methods of making loose particle, flake, or fiberbased large-surface-area, biocompatible, cell-culture-enhancing orbone-growth-enhancing surface having a nanotube covered structure,comprising: (a) applying a sodium hydroxide or potassium hydroxidechemical reaction with the particle material comprising Ti, Zr, Hf, Nb,Ta, Mo or W metal material, or a Ti, Zr, Hf, Nb, Ta, Mo or W alloy, toform a sodium titanate or related nanotube or nanofiber array structureon the particle surface; (b) providing hydrothermal treatment to convertthe sodium titanate or related nanotube or nanofiber array into oxidenanotube array; and (c) heat treating to convert amorphous nanotube ornanofiber into crystalline structure.

The invention provides methods of making loose particle, flake, or fiberbased large-surface-area, biocompatible, cell-culture-enhancing orbone-growth-enhancing surface having a nanotube covered structure,comprising: (a) by selecting a sheet, ribbon or wire material comprisingTi, Zr, Hf, Nb, Ta, Mo or W metal material, a Ti, Zr, Hf, Nb, Ta, Mo orW alloy, Ti, Zr, Hf, Nb, Ta, Mo or W oxide; (b) anodizing the refractorymetal wire or sheet to form a partially or fully penetrating metal oxidenanotube structure, optionally crystallizing the oxide to anatase orrutile phase; and (c) breaking up the oxidized material into loosepowders or fibers, optionally filling the nanotube pores with biologicalagents for accelerated cell or bone growth, or for therapeutic drugrelease purpose.

The invention provides nano-patterned, see-through cell culturesubstrate structures comprising a transparent thermosetting polymer,transparent thermoplastic polymer, transparent UV-light-curable polymer,or transparent glass base which is covered or coated with an opticallytransparent or translucent, and very thin film of material comprisingTi, Zr, Hf, Nb, Ta, Mo or W metal material, a Ti, Zr, Hf, Nb, Ta, Mo orW alloy, a Ti, Zr, Hf, Nb, Ta, Mo or W oxide, or Ti, Zr, Hf, Nb, Ta, Moor W nitride. In alternative aspects of the nano-patterned, see-throughcell culture substrate structures, the nano-patterned or micro-patternedsurface microstructure of the substrate has either pillars, tubes,lines, or pores with approximately 10 nm to approximately 500 μm featuresize; or, the nano-pattern is periodic or the nanopattern is random insize, shape or distribution; or the thermosetting polymer is selectedfrom polydimethylsiloxane (PDMS), polymethyl methacrylate (PMMA),melamine, Bakelite and epoxy resins; or the said thermoplastic polymeris selected from polyethene, polypropene, polystyrene, or poly vinylchloride; or the UV-curable polymer is selected frompolydimethylsiloxane (PDMS) and polymethyl methacrylate (PMMA); or theoptical transparency is at least 20%, or at least 40%, of the light sentthrough the substrate; or the thicknesses (thinnesses) of thetransparent or translucent coatings (e.g., Ti or TiO₂ related alloys,oxides, etc) is in a range of about 1 to 50 nm.

The invention provides methods of fabricating the nano-patterned,see-through cell culture substrate of claim 43, comprising the step ofimprinting using a stamp with pre-patterned surface, or nanoscalesurface etching using solvents or chemicals. In alternative aspects, thenano-patterning is carried out by a method comprising permanentmechanical impressing of a nano-patterned stamp on soft substratematerial selected from the group consisting of uncured thermosettingpolymer, heated and softened thermoplastic polymer, or heated andsoftened glass; or the patterning is performed by a method comprisingwet resist pattern transfer onto a transparent cell culture substrateusing a surface-patterned stamp followed by curing and chemical orreactive ion etching of the cell culture substrate through patternedresist layer and optional removal of the resist layer; or the patterningis performed by a method comprising impressing of a stamp into acontinuous layer of liquid resist film coating on a transparent plasticor glass followed by curing and chemical or reactive ion etching throughpatterned resist layer and optional removal of the resist layer; or thecuring of wet, patterned polymer is performed by a method comprisingthermal heat curing while it is being impressed by a nano or microstamp;or the curing of wet, patterned polymer is performed by a methodcomprising UV light illumination curing while it is being impressed by anano or microstamp; or the coating of an inorganic film of Ti, Ti oxide,Ti nitride and related refractory metals, oxides or nitrides on surfacenano-patterned transparent substrate is carried out by a methodcomprising physical or chemical means including sputtering, evaporation,atomic layer deposition, chemical vapor deposition, electroless platingor electroplating.

The invention provides methods of making a biocompatible andcell-growth-enhancing culture substrate comprising an elasticallycompliant protruding nanostructure substrate coated with Ti, TiO₂ orrelated metal and metal oxide films, comprising: (a) providing a surfacenanopatterned stamp; (b) impressing into a wet, uncured elastomer layerwith the nanostamp; (c) curing the polymer while being impressed bythermal curing or UV light curing; (d) releasing and removing the stamp;(e) depositing a thin film of Ti, Zr, Hf, Nb, Ta, Mo or W metalmaterial, a Ti, Zr, Hf, Nb, Ta, Mo or W alloy, a Ti, Zr, Hf, Nb, Ta, Moor W oxide, or Ti, Zr, Hf, Nb, Ta, Mo or W nitride by a methodcomprising a physical or chemical thin film deposition method. Inalternative aspects, the elastically compliant protruding nanostructurehas a configuration of a periodic or a random array of nanopillars,nanoballs, nanolines or nanomesh elements, or a combination thereof.

The details of one or more embodiments of the invention are set forth inthe accompanying drawings and the description below. Other features,objects, and advantages of the invention will be apparent from thedescription and drawings, and from the claims.

All publications, patents, patent applications, GenBank sequences andATCC deposits, cited herein are hereby expressly incorporated byreference for all purposes.

BRIEF DESCRIPTION OF THE DRAWINGS

The following drawings are illustrative of embodiments of the inventionand are not meant to limit the scope of the invention as encompassed bythe claims. The advantages, nature and additional features of theinvention will appear more fully upon consideration of the illustrativeembodiments described in the accompanying drawings. In the drawings:

FIG. 1A and FIG. 1B schematically illustrates exemplary configurationsof an extended and protruding, wire-like biocompatible structure forimproved toughness, strength, and mechanical locking of bone growtharound compliant, hairy-shaped or mesh-screen-shaped Ti (or Ti alloy),or any biocompatible alloy such as stainless steel, attached on the Tiimplant surface (functions, e.g., like reinforced concrete). FIG. 1aillustrates a hairy or gauze Ti wire mesh; FIG. 1b illustrates thestrongly locked in bone growth possible around the compliant, hairy ormesh screen structures of this invention.

FIG. 2 illustrates exemplary TiO₂ nanotube or nanopore arrays on thesurfaces of protruding hairy-shaped or mesh-screen-shaped Ti elementsbonded onto the Ti implant surface, and optionally the biological agentscan be stored in the nanotubes or nanopores for growth factor, drugdelivery, etc. The bonded regions of the protruding structures areillustrated.

FIG. 3A and FIG. 3B schematically illustrates exemplary self-organizedTiO₂ nanotube arrays grown on titanium substrate, which accelerates cellproliferation, e.g., as in accelerated cell proliferation on exemplaryTiO₂ nanotubes or nanopores. FIG. 3a illustrates a cell adhered andgrowing on an exemplary nanotube structure of this invention; FIG. 3billustrates a cell adhered and growing on an exemplary nanoporestructure of this invention.

FIG. 4A and FIG. 4B illustrates pictures of an exemplary microstructureof the vertically aligned TiO₂ nanotubes on titanium substrate, asillustrated by FIG. 4(a) scanning electron microscope (SEM) micrograph,FIG. 4(b) cross-sectional transmission electron microscope (TEM)micrograph.

FIG. 5A, FIG. 5B, FIG. 5C and FIG. 5D illustrates an exemplary processof diffusional bonding of hairy Ti or mesh-screen Ti onto the Ti implantbase using Ti film deposition followed by high temperature annealing; orhairy or mesh-screen material of any biocompatible alloy such asstainless steel, bonded on any biocompatible alloy such as Ti, Zr, Hf,stainless steel, etc. FIG. 5a shows an exemplary protruding structurecomprising hairy or mesh screen Ti wire on an implant; FIG. 5b showsanchoring thick film Ti deposit (1-2000 micrometer thick), optionallyoblique incidence plus rotating substrate; FIG. 5c shows an exemplarydiffusion annealed and bonded Ti layer (at 500-1300.degree. C./0.1-100hrs); FIG. 5d shows both the Ti wire surface and flat Ti surface areanodized to have TiO₂ nanotube or nanopore structure.

FIG. 6A, FIG. 6B, FIG. 6C and FIG. 6D schematically illustratesexemplary and various melt-bonding techniques for attaching protrudinghairy-shaped or mesh-screen-shaped Ti onto the surface of Ti implant, orto any biocompatible alloy such as stainless steel, ceramic, etc., andmelt-bonded onto the Ti implant. FIG. 6a shows an exemplary methodcomprising use of an induction-heating RF wave, plasma heating, e-beamheating, laser heating, torch heating and/or furnace heating; FIG. 6bshows the melt-bonded contact region between the mesh and substrate;FIG. 6c shows exemplary TiO₂ nanotubes on the wire surface byanodization; FIG. 6c shows exemplary recessed nanopores with TiO₂surface on the Ti wire surface.

FIG. 7A, FIG. 7B, FIG. 7C and FIG. 7D depicts exemplary hairy Ti ormesh-screen Ti (or any biocompatible alloy such as stainless steel)spot-welded onto the Ti implant. FIG. 7a shows an exemplary method usinga spot welding upper electrode in the shape of disk, plate, grid,vertical rod array, frame; in one aspect, the contact region is a noblemetal, e.g., Au, Pt, Pd or alloys; FIG. 7b shows an exemplary spotwelded Ti region; FIG. 7c shows exemplary TiO₂ nanotubes on the wiresurface by anodization; FIG. 7d shows exemplary recessed nanopores withTiO₂ surface on the Ti wire surface.

FIG. 8A and FIG. 8B schematically illustrates a side view of anexemplary hairy or wire-mesh Ti (or alloy), or an exemplarybiocompatible alloy such as stainless steel, attached onto Ti implantsurface for enhanced toughness, strength, and mechanical locking of bonegrowth around implant; FIG. 8a shows exemplary methods comprisingspot-welded, or induction melting-bonded, e-beam bonded, laser-bonded orbraze-bonded Ti wire mesh (single layer or multi-layer), having surfaceTiO₂ nanopore or nanotube array; FIG. 8b shows a strongly locked-in bone(or cell) growth around an exemplary woven or compliant, gauze Ti wiremesh structure of this invention.

FIG. 9A and FIG. 9B schematically illustrates an exemplary flat Tiimplant vs dual-structured, bone-locking Ti implant with attached hairyTi or Ti mesh screen to further enhanced, local adhesion and mechanicallocking; FIG. 9a shows an exemplary bonded protruding hairy wire or meshscreen Ti with surface nanopore or nanotube TiO₂; FIG. 9b shows anexemplary hairy or mesh-screen Ti wire mesh with surface nanopore ornanotube TiO₂.

FIG. 10A, FIG. 10B and FIG. 10C illustrates an exemplary monolayered Ti(or alloy) particles or fiber arrays attached onto Ti implant surfacefor enhanced toughness, strength, and mechanical locking of bone growtharound the particles; FIG. 10a shows an exemplary protruding Ti micro ormacro particles (or a cross-sectional view of fibers) attached on Tiimplant surface, by induction melt-bonding, e-beam melt bonding, laserbonding, spot-welding, braze-bonding, etc., and Ti implant with flat,round or curved surface Ti implant; FIG. 10b shows an exemplary surfacemodified Ti particles or fibers (and also the surface of the implantbase itself) with anodization-induced TiO2 nanotube or nanopore surface;FIG. 10c shows an exemplary nanoscale+microscale structure with stronglylocked-in bone growth around Ti particles or fibers.

FIG. 11 illustrates an exemplary Ti (or alloy) particle or fiberaggregate sintered and attached onto Ti implant surface for enhancedtoughness, strength, and mechanical locking of bone growth around theparticle aggregate; protruding, surface modified Ti particle aggregateor fiber aggregate, with anodization-induced TiO2 nanotube or nanoporeson the particle or fiber surface is shown on an exemplary Ti implantwith flat, round or curved surface Ti implant.

FIG. 12A and FIG. 12B schematically illustrates an exemplary magneticremote controllable drug delivery system based on densely spaced Tiparticles or wire mesh screen bonded onto Ti implant surface or anymaterial surface, such as metal, ceramic or polymer; FIG. 12a shows anexemplary surface modified Ti particle aggregate withanodization-induced TiO2 nanotube or nanopores on the particle surface,and an exemplary drug or biological agent (such as antibiotics,chemotherapy medicine, anti-stenosis drug, insulin, DNA, hormone, growthfactor, etc.) inserted into the nanopores, with a Ti support of implantwith flat, round or curved surface; FIG. 12b shows an exemplary surfacemodified Ti particle aggregate with anodization-induced TiO2 nanotube ornanopores on the particle surface with magnetic nanoparticles and drugor biological agent, and a Ti support or implant with flat, round orcurved surface.

FIG. 13A and FIG. 13B schematically depicts an exemplary magnetic remotecontrollable drug delivery system based on mesoporous aggregate materialfilled with magnetic nanoparticles; FIG. 13a shows an exemplary use ofmesoporous carbon, mesoporous silicon, mesoporous metal, mesoporousceramic or a mesoporous polymer aggregate; and a biocompatible substratesuch as Ti, inert metal, ceramic, polymer, any material coated withbiocompatible surface layer; and FIG. 13b shows an infiltrated (e.g., adrug or biological agent+magnetic nanoparticles), e.g., by an exemplarymethod comprising supercritical fluid deposition, or a boiled liquidmethod.

FIG. 14A, FIG. 14B and FIG. 14C schematically illustrates an exemplarydrug delivery system with nanowire, micro-wire or micro-ribbon arraythat holds a drug or biological agent and releases it by remotelyactivated magnetic field; FIG. 14a shows an exemplary actuate-ablenanowire (such as carbon nanotube forest coated with magnetic material),magnetic nano- or micro-ribbon, or magnetic nano- or micro-wire; FIG.14b shows how capillary-trapped drug or biological agent is released byinduced movement of magnetic wires or ribbons, and how the magnetic wireor ribbon is actuated to move by a remote magnetic field, e.g., aregular, sequential or gradient field applied; and FIG. 14c shows anexemplary device of the invention comprising a non-magnetic wire orribbon forest, e.g., aligned or partially tangled, comprising acapillary-trapped drug or biological agent, and how magneticnanoparticles can make the drug solution be released by a field-inducedmagnetic particle movement or by a high-frequency magnetic field heatingof particles, and the movement inducing the release of the compound inthe liquid (e.g., release of a drug-containing liquid).

FIG. 15A, FIG. 15B, FIG. 15C and FIG. 15D illustrates an exemplarymagnetic remote controllable drug delivery system based on directionallyformed porous material filled with magnetic nanoparticles; FIG. 15a ismeant to illustrate how a base material can be etched to formdirectional nano or micro-pores, where the base material can be, e.g.,Al, Si, ceramics, other metals or a polymer, or it can be a single phasematerial, or alternatively a two-phase or composite material for ease ofselective etching; FIG. 15b illustrates exemplary vertical pores formedin the base, e.g., a ceramic, Si, metal/alloy, or polymer material,prepared by chemical or electrochemical etching, thermal or plasmaetching, utilizing differential melting point or differential vaporpressure of component phases, differential sputter etch rate or ion etchrate (crystal orientation dependent or two phase'scomposition-dependent), or post-thermal chemical etching of melttextured (directional solidified) structure by induction, laser ore-beam melting, or sputter/resputter process. Optionally, abiocompatible coating can be applied; FIG. 15c illustrate the optionaluse of partial capping to reduce the pore entrance size, e.g., byoblique incidence sputtering or evaporation, quick electroplating, quickelectroless plating, or quick dipping in adhesives; FIG. 15d illustrateshow drugs or biological agents can be present in aqueous solutions, asdissolved or as colloidal solutions, and optionally, also comprisingmagnetic nanoparticles for remote-actuated drug delivery.

FIG. 16A, FIG. 16B and FIG. 16C schematically illustrates an exemplarymagnetic remote controllable drug delivery system based on porousstructures made by evaporated or sputtered thin or thick films, eitheras made or post-deposition-etched for removal of one of the phases; FIG.16a shows an exemplary porous Ti or TiO₂ surface made by evaporation, orby DC or RF sputtering, on a substrate; FIG. 16b shows this exemplaryproduct of the invention comprising a biological agent, such as a growthfactor, collagen, a hormone, a DNA or nucleic acid, etc.; FIG. 16c showshow magnetic or other movable functional particles and desiredcompositions, e.g., a drug, a DNA or nucleic acid, a growth factor, ahormones, etc., can be released by local heating, local magnetic field,electrical impulses for, e.g., controllable drug delivery, etc.

FIG. 17 schematically illustrates an exemplary, remote magneticallyactuated, on-off controllable or programmable drug release device usingoperational methods of moving or high-frequency heating of magneticparticles trapped in nanopores or micropores in the drug delivery systemimplants.

FIG. 18A, FIG. 18B, FIG. 18C, and FIG. 18D depicts exemplary structuresof elastically compliant implant material for bone growth using Ti basedmetal or alloy or any biocompatible alloy such as stainless steel; FIG.18a shows an exemplary Ti or Ti alloy implant (flat, round or curvedsurface), optionally with TiO₂ nanotube or nanopore surface; and anexemplary compliant, springy, or bent Ti wire, ribbon and/or mesh screenof pure metal or alloy, with TiO₂ nanotube or nanopore surface (e.g.,made by oblique incident evaporation, sputtering, or welding, brazing,induction-melt-bonding, e-beam melt-bonding, laser-melt-bonding, spotwelding, etc), and optionally comprising TiO₂ nanotube or nanoporesurface on the Ti wire surface, and optionally comprisingspacer/protector for abrasive insertion of Ti implants (e.g., screw-likeimplants into bones); FIG. 18b shows an exemplary Ti mesh screen bondedonto Ti implant; FIG. 18c shows an exemplary method comprising applyingpressure and electric voltage to the spot welder electrode array, andthe spot welder electrode array, in this example generating bent,springy Ti wires; and FIG. 18d shows an exemplary spot welder electrodearray in this example generating a Ti mesh screen bonded onto Tiimplant.

FIG. 19A and FIG. 19B schematically illustrates exemplary bone growthsteps around compliant implant material using Ti based metal or alloy orany biocompatible alloy such as stainless steel; FIG. 19a showing anintermediate stage of bone growth around an exemplary compliant Tispring implant, with compliant Ti wires or ribbons with TiO₂ nanotube ornanopore surface; and an optional spacer/protector for abrasiveinsertion of Ti implants (e.g., screw-like implants); and FIG. 19b showsan exemplary product of the invention comprising ribbons with TiO₂nanotube or nanopore surface; the compliant Ti serves as reinforcementas in a reinforced concrete.

FIG. 20A, FIG. 20B and FIG. 20C illustrates an exemplary fabricationmethod for converting the surface of a non-Ti type nanoporous ormicroporous materials to cell- or bone-growth-accelerating structure bythin biocompatible surface coating of Ti, TiO₂ or other relatedbiocompatible metals and alloys; FIG. 20a shows an exemplary product ofthe invention comprising non-Ti type, nanoporous or microporousmaterials (e.g., anodized Al₂O₃ membrane, porous Si, porous polymer),including nanopores or micropores, which optionally can be between about20 to 2000 nm diameter; FIG. 20b shows an exemplary Ti, TiO₂ typecoating, e.g., with 5 to 100 nm thick layer thick coating by sputtering,evaporation, chemical vapor deposition; FIG. 20c shows how cells orbones are grown in an accelerated manner on this exemplary Ti- orTiO₂-type coated nanopore structure.

FIG. 21A, FIG. 21B, FIG. 21C, and FIG. 21D schematically illustrates anexemplary process of creating a TiO₂ nanotube or nanopore surfacestructure on non-Ti type surfaces by thick Ti film deposition followedby anodization, including formation of TiO₂ nanotubes or nanopores byanodization of thick, deposited Ti on a non-Ti type substrate comprisingceramics, polymers, plastics, Si, Au, Pt and/or Al, etc.; FIG. 21a showsan exemplary product of the invention comprising non-Ti type substrate,e.g., comprising ceramics, polymers, plastics, Si, Au, Pt, Al., etc.;FIG. 21b shows an exemplary product with a configuration/shape made bymacro- or micro-shaping, e.g., by photolithography, machining, shadowmask polymer coating plus chemical etching, etc.; FIG. 21c shows anexemplary Ti type metal coating, e.g., with Ti, Zr, Hf, Nb, Ta, Mo, Wand their alloys among themselves or with other elements, by sputtering,evaporation, chemical vapor deposition, plasma spray, thermal spray,etc., FIG. 21d shows how cell- or bone-growth is accelerated by acoating of TiO₂ nanotubes or nanopores by anodization.

FIG. 22A describes the nature of cell- or bone-growth-acceleratingcoating of TiO₂ nanotubes or nanopores by anodization of thick-film Ticoating on an exemplary pre-patterned, non-Ti type substrate (ceramics,polymers, plastics, Si, Au, Pt, Al, etc.), and resultant cell or bonegrowth with a mechanically more reliable lock-in structure; FIG. 22Bshows how accelerated cell- or bone-growth is achieved on TiO₂ nanotubesor nanopores of the invention.

FIG. 23A, FIG. 23B and FIG. 23C illustrates exemplary bio implants forin vivo growth of bones, teeth, cells, organs, ex vivo functional biodevices such as artificial liver devices, orthopedic/dental implants, aswell as drug delivery devices and therapeutic devices based onbiocompatible implants; FIG. 23a shows various exemplary implants,including dental, periodontal, elbow, hip, knee and leg implants; FIG.23b shows exemplary implant comprising implanted cells or organs, or anexemplary artificial liver of this invention; FIG. 23b shows exemplarydrug-containing or drug delivery devices for, e.g., stents or otherblood vessels, and exemplary devices for, e.g., insulin, etc., ortherapeutic devices, e.g., for cancer treatment, etc.

FIG. 24 illustrates an exemplary accelerated cell growth devicecomprising an array of protruding, large-surface-area, hairy,mesh-screen or particulate shaped Ti members bonded onto Ti substrateand processed to have TiO₂ nanotube or nanopore surface, forapplications such as cell supply, rapid cell identification, harvestingof cell components, secreted proteins, albumin and other bio componentsgenerated by cultured cells; including illustrating exemplary arrays ofthe invention comprising protruding large-surface-area hairy,mesh-screen, or particulate shaped Ti members bonded onto Ti implantsurface and processed to exhibit TiO₂ nanotube or nanopore surface forrapid culture of cells, with at least one detection element, and with abiochip substrate.

FIG. 25 schematically illustrates an exemplary accelerated liver cellgrowth device comprising an array of protruding, large-surface-area,hairy, mesh-screen or particulate shaped Ti members bonded onto Tiimplant surface and processed to exhibit TiO₂ nanotube or nanoporesurface, for culturing a cell, e.g., a liver cell, optionally alsocomprising other cell types, for applications such as rapid toxicitytesting of drugs or chemicals, on a biochip substrate.

FIG. 26A, FIG. 26B and FIG. 26C schematically illustrates exemplaryembodiments of the inventive bio-chip test apparatus useful for drugtoxicity, chemical toxicity, or cell identification testing, with theapparatus comprising an array of the inventive, protruding,large-surface-area biomaterials, with the analytes detected by FIG.26(a) optical means, including use of any device for optical detection,e.g., using a microscope, fluorescent microscope, or CCD camera sensingof fluorescent or quantum dot tagged cells, and illustrating theaccelerated cell culture on these exemplary protruding,large-surface-area biomaterials; FIG. 26b shows an exemplary chemical orbiological analysis with a chemical or biological detection, e.g., basedon signature reactions; and FIG. 26c shows a magnetic sensor techniquefor magnetic sensor detection, e.g., by using magnetically targetedantibody, including use of a GMR or TMR sensors as a magnetic sensorarray.

FIG. 27 describes an exemplary method of creating loose,TiO₂-nanofiber-coated or TiO₂-nanotube-coated Ti powder (using, e.g.,spherical, elongated, random or short-wire shape particles) by rotatingor moving the electrode (alternatively, the bottom electrode can staystill but the powders are now agitated instead, and made to move aroundto occasionally touch the electrode); noting optionally: electrolytesat, e.g., HF, 0.2 to 2.0 wt % concentration, or alternatively at 0.5 to1 wt %), Ti powder or flake being anodized on contact, and a rotatingelectrode (a cathode).

FIG. 28 schematically illustrates an alternative technique of creatingTiO₂-nanofiber-coated or nanotube-coated Ti powder of micro-sized ormacro-sized (e.g., spherical, elongated, random or short-wire shape orflake-shape or needle particles, by NaOH chemical treatment to formsodium titanate intermediate phase nanotubes first on the Ti powdersurface, then converting them into TiO₂ nanofibers or nanotubes byhydrothermal treatment, followed by optional heat treatment to convertamorphous TiO₂ into crystallized TiO₂; and the exemplary steps of NaOHtreatment (to generate sodium titanate nanotubes/nanofiber),hydrothermal treatment (to generate Amorphous TiO₂ nanotubes/nanofiberson the surface), and optional annealing for crystallization to ananatase (Anatase TiO₂ nanotubes/nanofibers on the surface).

FIG. 29A, FIG. 29B and FIG. 29C schematically illustrates an exemplaryinventive process of utilizing an extended anodization to create thinnerand grindable TiO₂ wire or ribbon to produce powder, flake, short fiber,etc., each segment having a cell-growth-accelerating TiO₂ nanotube ornanopore surface structure; FIG. 29a illustrates use of Ti wire orsheet; FIG. 29b illustrates use of anodized Ti wire or sheet with mostlyTiO₂ surface+optional crystallization heat treatment; FIG. 29cillustrates use of ground, crushed or cut TiO₂ or related material (aTi, Zr, Hf, Nb, Ta, Mo and/or W metal material, a Ti, Zr, Hf, Nb, Ta, Moand/or W alloy, a Ti, Zr, Hf, Nb, Ta, Mo and/or W oxide or nitride,and/or stainless steel or ceramic) into powder, flake, short wire, etc.

FIG. 30 illustrates an exemplary use of TiO₂-nanotube-coated powders ormesh screen of TiO₂ (with optional Ti core) to accelerate the bonegrowth and regenerative-healing process; and use of an exemplary productof this invention comprising bone cement comprising TiO₂-surfaced loosetemplates (with optional Ti core) of microwire mesh, powder, flake, etc.with optional growth factors, nutrients, antibiotics, hormones, genes,hydroxyapatite, natural bone powder, bio-degradable polymer,bio-compatible or bio-inert bone cement, bio-active glass, magneticnanoparticles, etc.

FIG. 31 describes an exemplary dental use of TiO₂-nanotube-coatedpowders or mesh screen of TiO₂ (with optional Ti core) for accelerateddental bone growth; and use of an exemplary product of this inventionfor accelerated growth of regenerated dental bone on templates of looseTiO₂ (with optional Ti core) having nanotube or nanopore surface, in theconfiguration of microwire mesh, powder, flake, etc. with optionalgrowth factors, nutrients, antibiotics, hormones, genes, hydroxyapatite,natural bone powder, bio-degradable polymer, bio-compatible or bio-inertbone cement, magnetic nanoparticles, etc.

FIG. 32 schematically illustrates an exemplary periodontal use ofTiO₂-nanotube-coated powders or mesh screen of TiO₂ (with optional Ticore) to accelerate the periodontal tissue growth andregenerative-healing process; and use of an exemplary product of thisinvention for regenerating periodontal tissue on a template of TiO₂(with optional Ti core) in the form of microwire mesh, powder, flake,etc. with optional growth factors, nutrients, antibiotics, hormones,genes, hydroxyapatite, bio-degradable polymer, bio-compatible orbio-inert bone cement, etc.

FIG. 33A, FIG. 33B, FIG. 33C, and FIG. 33D schematically illustrates anexemplary nano or micro imprinting method to fabricate nano-patterned,completely or partially “see-through”, or transparent, cell culturesubstrates of this invention based on plastic or glass, e.g., a culturedish or equivalent, which are coated with a thin layer of Ti, TiO₂ orrelated metals or oxides (including, e.g., a Ti, Zr, Hf, Nb, Ta, Moand/or W metal; a Ti, Zr, Hf, Nb, Ta, Mo and/or W alloy; and/or, a Ti,Zr, Hf, Nb, Ta, Mo and/or W oxide or nitride, and/or stainless steel orceramic). FIG. 33a shows nano-imprint stamping, e.g., to generatepillars, lines or pores, having about a 10 nm to 500 Cpm feature size.FIG. 33a also shows as a top layer, a soft matrix (uncuredthermosetting) polymer, heated thermoplastic polymer, uncured glassprecursor, heat-softened glass, etc. and the bottom layer a supportstructure. FIG. 33a shows an exemplary nano-imprinting process at roomtemp or elevated temperature, which can be heat cured, UV cured,catalyst cured polymer and/or solidified by cooling, e.g., glass,thermoplastic polymer. FIG. 33c shows an exemplary nano-cavity,nanopillar or nano-grid, e.g., designed to be periodic or intentionallynon-periodic to minimize diffraction and interference of passing light.FIG. 33c shows an exemplary deposited thin film, e.g., as a Ti or TiO₂,or other bio-compatible metals, alloys, oxides, nitrides of Zr, Ta, Nb,Mo, Hf, Cr, and/or stainless steel or ceramic, and the like, depositedusing, e.g., oblique or vertical sputtering, evaporation, etc.

FIG. 34A and FIG. 34B compares two alternative nano-pattern embodimentsof the invention, periodic versus (vs) random nano or micro imprintedpatterns in a see-through cell culture substrate based on plastic orglass, coated with a thin layer of Ti, TiO₂ or related metals or oxidesor nitrides (including, e.g., a Ti, Zr, Hf, Nb, Ta, Mo and/or W metal; aTi, Zr, Hf, Nb, Ta, Mo and/or W alloy; and/or, a Ti, Zr, Hf, Nb, Ta, Moand/or W oxide or nitride, and/or stainless steel or ceramic). FIG. 33ashows an exemplary periodic nanopattern of Ti, TiO₂ or related metals oroxides-coated cell culture substrate. FIG. 33b shows an exemplaryrandom, or non-periodic nanopattern of Ti, TiO₂ or related metals ormetal oxide-coated cell culture substrate.

FIG. 35A and FIG. 35B schematically illustrates an alternative method offabricating transparent or translucent cell-culture substrate by nano ormicro imprinting plus chemical or reactive ion etched (RIE) etchingmethod plus coating of a thin layer of Ti, TiO₂ or related metals oroxides or nitrides (including, e.g., a Ti, Zr, Hf, Nb, Ta, Mo and/or Wmetal; a Ti, Zr, Hf, Nb, Ta, Mo and/or W alloy; and/or, a Ti, Zr, Hf,Nb, Ta, Mo and/or W oxide or nitride, and/or stainless steel orceramic). This exemplary method comprises a nano imprint patterning of amold onto a “resist layer” of a transparent substrate, to etch-patternthe transparent substrate (e.g., glass or polymer substrate) through theresist layer. As shown in FIG. 35a , the liquid resist layer can becured by heat or UV light, or impressed on thermoplastic solid polymer.As shown in FIG. 35b , alternatively, one can continue an RIE Etch orapply chemical etch for substrate patterning; then one can remove theresist later and deposit thin, almost or completely transparent Ti, TiO₂or related metals or oxides coating on the transparent substrate.

FIG. 36A, FIG. 36B, FIG. 36C, FIG. 36D and FIG. 36E illustrates analternative method of fabricating transparent or translucentcell-culture substrate by nano or micro imprint based resist patterntransfer followed by chemical or RIE etching plus coating of a thinlayer of Ti, TiO₂ or related metals or oxides or nitrides (including,e.g., a Ti, Zr, Hf, Nb, Ta, Mo and/or W metal; a Ti, Zr, Hf, Nb, Ta, Moand/or W alloy; and/or, a Ti, Zr, Hf, Nb, Ta, Mo and/or W oxide ornitride, and/or stainless steel or ceramic). In the embodiment of FIG.36a , the resist pattern is made, or transferred, with an imprint stampfor patterning. FIG. 36 b shows an exemplary ink transfer by animprinting process. FIG. 36c shows an exemplary patterned ink, nano- ormicro-patterned “resist” array. FIG. 36d shows an exemplary protrudingnanopillar or recessed nanopore pattern made by chemical or RIE etch.FIG. 36e shows an exemplary process depositing thin film Ti, TiO₂ orrelated metals or oxides, including nitrides and the like.

FIG. 37 illustrates a picture of an exemplary silicon nano imprint stampcontaining 25 nm dia. periodic islands.

FIG. 38A, FIG. 38B, FIG. 38C, FIG. 38D and FIG. 38E is a schematicillustration of an exemplary polymer-based cell culture substrate of theinvention cured while imprinted using UV light or heat, and coated witha thin layer of Ti or TiO₂, or a Ti, Zr, Hf, Nb, Ta, Mo and/or W metal;a Ti, Zr, Hf, Nb, Ta, Mo and/or W alloy; and/or, a Ti, Zr, Hf, Nb, Ta,Mo and/or W oxide or nitride, and/or stainless steel or ceramic. Thisembodiment can alternative use from between about 20 nm to 20 μm featurenano stamps or micro stamps, which can be imprinted on spin-coated,uncured polymer, e.g., heat curable or UV light curable PDMS(polydimethylsiloxane), and cured while being imprinted. FIG. 38a showsan exemplary method for imprinting a “nano” stamp on a wet, uncuredpolymer resist layer, e.g., a UV-curable or a heat-curable layer, e.g.,PDMS. FIG. 38b shows an exemplary method to UV-cure, e.g., by UV lightillumination through transparent substrate or by heating. FIG. 38c showsstamp release to obtain a nano-imprinted polymer pattern. FIG. 38d showsthe results of the additional step of coating with, e.g., a thin layerof Ti or TiO₂, or a Ti, Zr, Hf, Nb, Ta, Mo and/or W metal; a Ti, Zr, Hf,Nb, Ta, Mo and/or W alloy; and/or, a Ti, Zr, Hf, Nb, Ta, Mo and/or Woxide or nitride, and/or stainless steel or ceramic. FIG. 38e shows thesubstrate after peeling off the support structure.

FIG. 39 illustrates a scanning electron microstructure of an exemplarynano-imprinted pattern on a PDMS (polydimethylsiloxane); thisillustrates an exemplary nano-imprinted pattern on PDMS(Polydimethylsiloxane) at 200 nm diameter periodic pores, approximately100 nm deep features.

FIG. 40 illustrates an SEM micrograph showing an exemplarynano-patterned PDMS (polydimethylsiloxane) cell-culture substrate withprotruding array of soft and compliant balls of about 200 nm diameter.

It is to be understood that the drawings are for purposes ofillustrating the concepts of the invention and are not to scale.

DETAILED DESCRIPTION

The present invention provides biocompatible nanostructures withsignificantly increased surface area, such as products of manufacturecomprising nanotube and/or nanopore arrays on the surface of metallic,ceramic, or polymer materials for e.g., enhanced cell and bone growth,for in vitro and in vivo testing, cleansing reaction, implants andtherapeutics, e.g., as drug delivery devices. In one aspect, theinvention provides products of manufacture comprising biomaterialscomprising Ti oxide type nanostructures with various protruding andextended biomaterial configurations; where these biomaterialconfigurations can enable accelerated cell growth and can be useful for,e.g., rapid acting and secure orthopedic, dental, periodontal,cell/organ implants, therapeutics, disease diagnostic, drug toxicitytesting, and cell supply applications.

Exemplary substrate biomaterials or surfaces of the substratebiomaterials of the invention can comprise Ti and Ti oxide as well asalloys containing Ti or Ti oxide by at least 50%, 51%, 52%, 53%, 54%,55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%,69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%,83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99%, or more or more weight %. Other related materials such asZr, Hf, Nb, Ta, Mo, W, and their oxides, or alloys of these metals andoxides by at least 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%,60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%,74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%,88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more ormore weight % can also be used.

The structures of the invention can be used with any TiO₂ nanotubulearray structure, e.g., as described in PCT Patent Application#PCT/US2006/016471, filed on Apr. 28, 2006, Jin et al. Using thestructures of the invention can provide enhanced cell and bone growth,or can provide improved Ti or TiO₂ configurations in nanopore ornanotube configurations. The nanostructures of the invention cancomprise Ti or TiO₂ or equivalent structures made of other materials butcoated with a biocompatible Ti or TiO₂ film. The structures of theinvention can allow enhanced cell adhesion and accelerated growth, forexample by at least 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%,60%, 61%, 62%, 63%, 64%, 650%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%,74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%,88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more or100% or faster, and or in another aspect, at least 120%, 150% or 200% orfaster.

This invention provides novel, biocompatible nanostructuredbiomaterials, devices comprising such biomaterials, and fabricationmethods thereof. The biomaterials of the invention can have a variety ofadvantageous structures and surface configurations, as discussed herein:

1. Biomaterials with Strongly Bonded, Protruding Features:

The invention provides products of manufacture/compositions comprisingmacroscopically or microscopically extended biomaterial topography,which can provide a lock-in mechanical integrity at a implant-hardtissue interface; and in one aspect, providing a TiO₂ nanotube type nanostructure comprising on its surface one or more protrusion features(e.g., biocompatible surfaces comprising structures, as describedherein), and in one aspect, the surface of the base implant provides adesirable cell- or bone-growth-accelerating characteristics.

2. Externally and Remotely Controllable Drug-Delivery Systems:

The invention provides products of manufacture/compositions comprisingnanotube or nanopore arrays, or micro-wires or micro-ribbon arrays,e.g., on implant surface, which in alternative aspects are utilized as areservoir for drug and other biological agents, which can haveadvantageous characteristics of magnetically actuated, on-demand drugrelease capability.

3. Elastically Compliant Implant Material for Bone Growth:

The invention provides products of manufacture/compositions comprisingsubdivided, spring-like fiber or mesh screen shape implants forstress-accommodation and minimal separation failures at the implant-hardtissue interface, which in alternative aspects provides strength andtoughness reinforcement of the grown bone via bone-metal wire compositeformation.

4. Non-Metallic or Non-Ti Based Substrates the Surfaces of which havebeen Converted to TiO₂ Type Nanotubes or Nanopores:

The invention provides products of manufacture/compositions comprising athin film coating of Ti and/or TiO₂, which can be applied onto thesurfaces of already nanoporous material, and/or a thick film Ti isdeposited and anodized to create TiO₂ nanotube type, which inalternative aspects can exhibit desirable cell or bone growthaccelerating characteristics.

5. Biocompatible Materials Configured in Loose Particles, LooseShort-Fibers, or Loose Flakes:

The invention provides products of manufacture/compositions comprisingpowder surfaces processed to comprise nanopore or nanotube arraynanostructure, so that the loose powders exhibit cell- orbone-growth-accelerating characteristics, which in alternative aspectscan be useful for bone cement and other tissue connection applications.

The invention provides products of manufacture/compositions comprisingoptically transparent or translucent, surface-nanostructured polymer,plastic or glass substrates, with coating comprising Ti, Ti oxide, Tinitride or related refractory metal, oxide or nitride films; and inalternative aspects, also comprising cell-growth-enhancing culturesubstrates comprising elastically compliant protruding nanostructuresubstrates coated with, in alternative aspects, Ti, TiO₂ or relatedmetal and metal oxide films.

1. Biomaterials with Strongly Bonded, Protruding Features

One exemplary embodiment comprises a solid substrate of biocompatiblematerial which is macroscopically or microscopically extended in spacewith protruding, strongly and permanently attached, three-dimensional,high-surface-area wire array, mesh screen array, or particle-assemblyarray. Another exemplary embodiment comprises having the surface of eachof these attached wires, mesh screens, or particles processed to have anarray of TiO2 nanotube type or nanopore type nanostructure on thesurfaces of these protrusion features; and in one embodiment, thehigh-surface-area wire arrays, mesh screen arrays, or particle-assemblyarrays are also on the surfaces of the base implant material itself soas to provide cell- or bone-growth-accelerating characteristics.

In one aspect, these configurations provide large-surface-areabiocompatible materials not only from the surface nano-features such astitanium oxide nanotubes or nanopores but the protruding extendedthree-dimensional structures. The increased overall surface areastogether with the conditions of secure cell or bone adhesion at theimplant-tissue interface allow accelerated and viable cell growth andbone growth, with significantly enhanced mechanical bond strength due tothe extended structure. Optionally, growth factors and other biologicalagents are added and stored in the nanotubes or nanopores formultifunctional advantages and for even further accelerated growth ofhealthy cells.

Shown schematically in FIG. 1(a) is an exemplary configuration of anextended and protruding, wire-like biocompatible structure for improvedtoughness, strength, and mechanical locking of bone growth aroundcompliant, hairy-shaped, or mesh-screen-shaped Ti (or Ti alloy such asTi—V—Al), or any biocompatible alloy such as metals and alloyscomprising Zr, Hf, Nb, Ta, Mo, W, or stainless steel, attached on the Tiimplant surface. The desired shape of the extended and protruding,wire-like biocompatible structure can be an array of isolated wires withtheir arrangement in vertical, inclined, or random orientation. Thewires or fibers can be either straight, curved or bent. Alternatively,instead of isolated wires, they can be mutually connected, e.g., in theform of mesh screen, woven screen, or gauge shape.

The protruding part of the structure can be pre-assembled (e.g., in amesh-screen shape) and then bonded onto the substrate by various methodssuch as diffusion bonding, or partial melt bonding, such as by using ase-beam, laser, DC or RF plasma heating, or RF (radio frequency)induction heating, or electrical spot welding as illustrated in FIGS.5-7. Alternative methods of preparing such a protrusion featureillustrated in FIG. 1(a) are not excluded, for example, a nearlyparallel array of vertical wires or microwires of Ti (or other metalsand alloys comprising Zr, Hf, Nb, Ta, Mo, W) can be brought downsimultaneously and in a parallel manner onto the Ti implant substratefor physical contacts, the tips in contact with the substrate areelectric-arc-bonded, then the extra length of the wires are sheared offto leave a forest of short wires attached onto the substrate.

Another alternative process consists of simple dropping of micro ormacro fibers of Ti (or Ti-alloy, stainless steel or other biocompatiblemetallic materials, as described herein) onto the implant surface, andbond them onto the implant surface under compression, e.g., by utilizingelectric arc spot welding or heating to high temperature for diffusionbonding. Such bonding processes for loose Ti fibers can be done even ona non-flat implant surfaces by utilizing mechanically adaptivecompression unit. For heating and diffusion bonding of Ti and relatedmetals or alloys onto the implant surface; exemplary heat treatingatmosphere are either inert or reducing atmosphere, such as Ar, He, orH₂ containing gas atmosphere, or vacuum atmosphere.

The implants with the bonded protruding features of FIG. 1 (e.g., Tiimplant with bonded, protruding Ti mesh screen) are thenelectrochemically anodized to produce a surface TiO₂ nanotube arraystructure to impart accelerated cell- or bone-growth characteristics onthe surface of the protruding structure as well as the surface of thebase implant material.

The implants with the protruding structure is then optionally heattreated to convert the generally amorphous TiO₂ material into acrystalline phase can be into the anatase TiO₂ phase, but not excludingother phases such as the rutile phase. The desired heat treatmentconditions including annealing at 350-600.degree. C. for 0.1-10 hr, orin another aspect, 450-550.degree. C. for 0.5-5 hr. The heating rate hasto be carefully chosen as slower than 5.degree. C./hr so that crumblingof the crystallizing phase is prevented. Such a heat treatment alsorelieves much of the mechanical residual stresses that might have beenintroduced during the bonding of the protruding structure, thusimproving the fracture toughness and fatigue life of the bone or teethimplants.

The desired diameter of the wires composing the protruding structure canin one aspect be in the range of 10-10,000 micrometers, or in anotheraspect, in the range of 25-500 micrometers. The desired thickness of theprotruding structure depends on specific applications and the averagediameter of the wires or fibers involved. In one aspect, the desiredoverall thickness of the protruding structure layer is 0.01-10 mm, or inanother aspect, 0.05-2 mm.

Shown schematically in FIG. 1(b) is the locked-in bone growth around theprotruding, hairy or mesh screen Ti with TiO₂ nanotube array surfacestructure for accelerated cell- or bone-growth characteristic. Such amechanically locked-in composite structure of the grown bone togetherwith the embedded implant protrusion ensures that the grown bone isstrongly bonded onto the base implant surface with a drasticallyminimized possibility of undesirable interface separation at thehard-tissue/implant interface.

The drawing in FIG. 2 shows an enlarged view of the FIG. 1 drawing. Itillustrates TiO₂ nanotube or nanopore arrays formed on the surfaces ofhairy-shaped or mesh-screen-shaped, protruding Ti elements bonded ontothe Ti implant surface. The TiO₂ nanotube or nanopore arrays can beformed using anodization processes described earlier, for example, seethe article by S. Oh et al., “Growth of Nano-scale Hydroxyapatite UsingChemically Treated Titanium Oxide Nanotubes”, Biomaterials (2005)26:4938-4943, and “Significantly Accelerated Osteoblast Cell Growth onAligned TiO₂ Nanotubes”, Journal of Biomedical Materials Research (2006)78A:97-103.

In one aspect, the pore spaces in the TiO₂ nanotubes or nanoporesillustrated in FIG. 2 can be utilized to store desirable biologicalagents such as biomolecular growth factors like BMP (bone morphogeneticprotein) or collagens, antibiotics, drug molecules, inorganicnanoparticles, etc. for steadily and passive supply to the in-vivo orin-vitro environment for further accelerated cell/bone growth or formedical therapeutics. The active, on-demand drug delivery utilizing acombination of such nanotubes/nanopores and inserted magnetic particleswill be describes later as a separate embodiment section.

The TiO₂ nanotube arrays or nanopore arrays grown on titanium substrateby anodization and other processes significantly enhance cell adhesion,and accelerate cell proliferation as illustrated in FIG. 3. A part ofthe reasons for such accelerated bio activity appears to be the enhancedadhesion of the growing cell front (filopodia) into the TiO₂ nanoporesat the early stage of cell growth and proliferation. See the article byS. Oh et al., “Significantly Accelerated Osteoblast Cell Growth onAligned TiO₂ Nanotubes”, Journal of Biomedical Materials Research (2006)78A:97-103.

The invention provides structures having a secure mechanical lock-in forgrowing cells or bones relative to the implant surface due to thepresence of the protruding structures, e.g., in the bioimplants of theinvention. A secure attachment ensures continued proliferation andintegration of healthy cells or bones. Depicted in FIG. 4 are theexemplary microstructures of the vertically aligned TiO₂ nanotubes onthe protruding Ti wire or mesh screen structure as well as the titaniumsubstrate itself. The microstructure of FIG. 4(a) represents a scanningelectron microscope (SEM) micrograph of the TiO₂ nanotube arrays, whileFIG. 4(b) represents a cross-sectional transmission electron microscope(TEM) micrograph of the TiO₂ nanotube arrays.

An exemplary process of the invention is to attach the protruding Tistructures in FIG. 1(a) and FIG. 2 to the Ti implant base to utilize aprocess of diffusional bonding as illustrated in FIG. 5. Whilediffusional bonding can be carried by simple contact and heating of twometals under compression, the invention calls for an addition ofphysical vapor deposited metal to enhance the diffusional bond strength.This exemplary process sequence, as illustrated in FIG. 5, can comprisefive basic steps:

i) Step 1—The protruding structure of hairy Ti or mesh-screen Ti isfirst prepared as a layer material and cut to pieces that match the sizeof the Ti implant base. The protruding structure is then placed on topof the implant base as illustrated in FIG. 5(a). The protrudingstructure is urged to physically contact the surface of the implant baseat as many contacts as possible by elastically compressing or gentlyclamping the mesh screen down during Ti thick film deposition, using agrid-shaped or a finger-shaped rod retainer structure (not shown in thefigure). The rods in the retainer structure can comprise a surfaceoxidized, metal rod material, so that the retainer rods do not stick tothe protruding mesh screen Ti structure during the subsequent diffusionbond process.

ii) Step 2—A thick film of Ti is then deposited onto the protrudingstructure and the implant base as illustrated in FIG. 5(b). The desiredthickness of the Ti anchoring film is in the range of 100-2000micrometer, which can be deposited either by physical vapor depositionsuch as sputtering or evaporation, or by chemical vapor deposition. Inorder to deposit the film relatively uniformly despite the interferenceof shadowing effect caused by the upper portion of the wire or meshscreen structure, a frequent rotation or tilting of the substrate duringthe film deposition in combination with an oblique incident depositioncan be used. With regard to the choice of deposited film material, theuse of the same material, e.g., Ti base protruding structure, Ti baseimplant, as well as Ti anchoring film can be used for ease andreliability of diffusional bonding without complications and unknowns,e.g., avoiding a possibility of forming a brittle intermetallic alloycompound. In addition to Ti, other alloys of Ti (such as Ti—Al—V), orany biocompatible metals or alloys such as Zr, Hf, Nb, Ta, Mo, W andtheir alloys, or stainless steels, may be utilized. Dissimilar metals oralloys, for example, bonding of stainless steel mesh screen on Tiimplant using Ti film deposition, can also be used; in one aspect,appropriate precautions in the selection of the matching alloycomposition and processing conditions can produce reliable bonding ofthe protruding structure.

iii) Step 3—The protruding structure and the base implant covered bydeposited thin film is then subjected to a high temperature annealingfor diffusional bonding of the Ti protruding structure onto the Tiimplant, with the deposited thick film of Ti serving as the anchoringmaterial, thus inducing a strong bonding attachment of the protrudingstructure onto the Ti implant base, as illustrated in FIG. 5(c). Thedesired diffusion annealing and bonding process includes heating andholding at a high temperature of between about 500 to 1300.degree. C.,or 500 to 1200.degree. C., 500 to 1100.degree. C., or 500 to1000.degree. C., for about 0.1 to 100 hrs, or in one aspect, in areducing atmosphere (such as hydrogen-containing gas atmosphere) or aninert atmosphere (such as argon or helium atmosphere) to minimizeoxidation of the Ti surface, which interferes with the intended metaldiffusional bonding. In one aspect, furnace heating is used, and it canbe simple, straightforward, and of low cost, however the use of otherheating techniques such as induction heating, e-beam heating, laserheating, and torch heating for the diffusion bonding is not excluded.

iv) Step 4—The bonded structure is then subjected to the anodizationprocess described earlier, so that both the surface of the Ti wire ormesh screen and the surface of the Ti implant base material are anodizedto have TiO₂ nanotube or nanopore structure for accelerated cell- orbone-growth. The invention also provides alternative processes such aschemical etching, surface melt evaporation, plasma etching, etc, insteadof anodizing, can also be utilized to induce the surface nanostructuresof metallic or oxide nanopores, nanotubes, nanowires on the hairy orwire-mesh protrusion structure. The space within each of the nanotubesor nanopores can also be utilized to store biological agents such asgrowth factors, antibiotics, genes, DNAs, therapeutic drugs, metallic ormagnetic nanoparticles, etc. to further accelerate the cell or bonegrowth, or to serve as an implanted medical therapeutic devices forapplications such as cancer treatment.

v) Step 5—The anodized assembly is optionally heat treated, e.g., nearthe temperature of about 500° C. for 0.1-10 hrs to crystallize thenanotube and obtain a desirable crystal structure such as the anatasephase.

FIG. 6 illustrates another exemplary process of the invention comprisingattaching the protruding Ti structures in FIG. 1(a) and FIG. 2, anon-contact heating process, to the Ti implant base is to utilize aprocess of melt bonding as illustrated in the figure. The expression“melt-bonding” used here is defined as having a broad meaning, whichincludes high temperature diffusion bonding as a result of suchnon-contact heating even if the overall metal temperature remains belowthe melting point of the hose substrate metal surface or the wire meshmetal and hence no melting occurs in a strict sense. This exemplaryprocess of the inventive, comprising melt bonding for biomaterialsfabrication, can comprise four steps:

i) Step 1—The protruding structure of hairy Ti or mesh-screen Ti in theoverall form of a layer material is cut to pieces that match the size ofthe Ti implant base, and is placed on top of the implant base asillustrated in FIG. 6(a). The structure is urged to physically contactthe surface of the implant base at as many contacts as possible byelastically compressing or gently clamping the mesh screen down using agrid-shaped or a finger-shaped rod retainer structure (not shown in thefigure). The rods in the retainer structure can comprise a surfaceoxidized, metal rod material so that they do not melt and stick to theprotruding mesh screen Ti structure during the subsequent melt-bondingheating process of FIG. 6(a).

ii) Step 2—This assembly structure is then subjected to the melt-bondingprocess to attach the protruding structure to the implant base, asillustrated in FIG. 6(b). The heating process can be carried out invacuum, inert atmosphere (e.g., using Ar or He) or in a reducingatmosphere (e.g., using hydrogen gas or a mixture of hydrogen and otherinert gases). Exemplary heating methods include induction heating usingRF electromagnetic field, e-beam heating, laser heating, torch heating,and furnace heating. Other methods of heating are not excluded. Withregard to the choice of materials involved, the use of the samematerial, e.g., Ti base protruding structure and Ti base implant can beused for ease and reliability of melt-bonding associated with the use ofthe same material. In addition to Ti, other alloys of Ti (such asTi—Al—V), or any biocompatible metals or alloys of Zr, Hf, Nb, Ta, Mo,W, or stainless steels, may be utilized. Dissimilar metals or alloys,for example, melt-bonding of stainless steel mesh screen on Ti implant,can also be used since appropriate precautions in the selection of thematching alloy composition and processing conditions can producereliable bonding of the protruding structure. The protruding structureand/or the Ti implant base are heated to a sufficiently high temperatureso that there is local surface melting (or surface softening anddiffusion bonding), for example, heating to a surface temperature of800-2000° C. for a general duration in the range of about 0.01-100minutes, or in another aspect, 0.1-10 minutes.

iii) Step 3—After the melt-bonding, the assembled structure is subjectedto the anodization process described earlier, so that both the surfaceof the Ti wire or mesh screen and the surface of the Ti implant basematerial are anodized to have TiO₂ nanotube or nanopore structure foraccelerated cell- or bone-growth as indicated in FIGS. 6(c) and (d).Other alternative processes such as chemical etching, surface meltevaporation, plasma etching, etc, instead of anodizing, can also beutilized to induce the surface nanostructures of metallic or oxidenanopores, nanotubes, nanowires on the hairy or wire-mesh protrusionstructure, and hence the use of such alternative processes is notexcluded. The space within each of the nanotubes or nanopores canoptionally be utilized to store biological agents such as growthfactors, antibiotics, genes, DNAs, therapeutic drugs, metallic ormagnetic nanoparticles, etc. to further accelerate the cell or bonegrowth, or to serve as an implanted therapeutic medical treatmentdevice.

iv) Step 4—The anodized assembly is optionally heat treated, e.g., nearthe temperature of about 500° C. for 0.1-10 hrs to crystallize thenanotube and obtain a desirable crystal structure such as the anatasephase.

FIG. 7 schematically shows an alternative exemplary method of theinvention comprising strongly bonding the protruding structure to theimplant base by spot welding. The protruding structure can behairy-shaped, fiber-shaped or mesh-screen-shaped Ti. Other similarlybiocompatible Ti-base alloys (e.g., Ti—Al—V alloys) or other refractorymetals (e.g., Zr, Hf, Nb, Ta, Mo, W and their alloys), or stainlesssteels can also be used for the protruding structure as well as for theimplant base.

The process of spot welding is well established in the engineeringfield. It is a type of resistance welding used to attach thin pieces ofmetal or alloy parts. It uses two large electrodes which are placed oneither side of the surface to be welded, and passes A large electricalcurrent is passed through the metal parts involved and heats up themetal contact area The degree of heating near the contact area isdetermined by the amplitude and duration of the current used. Metalswith higher electrical and thermal conductivity generally require largerelectrical currents to obtain a comparable heating effect.

As the contact of the hairy or mesh-screen protruding structure with thebase implant occurs at isolated spots, the spot welding is an efficientapproach of bonding such a structure. Referring to FIG. 7(a), one of thespot welding electrode is placed underneath the implant base materialwhile the upper electrode compresses down on the protruding structure asillustrated in FIG. 7(a). The shape of the spot welding upper electrodecan be a disk, plate, grid, vertical rod array, or frame. A disk- orplate-shaped electrode contacts and presses down on most of the topportion of the hairy or mesh-screen shaped protruding structure.Alternatively, if the mesh screen is highly porous, a finger-shape (avertical rod array) or linear grid-shape electrode can be prepared andinserted into the middle or lower portion of the mesh screen. Theduration of spot welding can be in the range of about 0.1 to 5 seconds.The spot welding process can be repeated if necessary, either on thesame spot or at nearby different spots.

In one aspect of the invention, the electrode materials (both the upperand lower electrodes) have to be carefully selected in order to avoidcontamination by the commonly used electrode material such as a Cuelectrode, the surface of which can be locally melted and alloyed ontothe surface of the Ti implant and the Ti protruding structure duringspot welding. As copper is not necessarily considered a fullybiocompatible material, the invention calls for a preferable selectionof the electrode material (at least near the electrode surface), amonghigh electrical conductivity noble metals and alloys such as Au, Pt, Pd,and their alloys. The use of Ti, W, or other refractory metal electrodesis not excluded.

After the spot welding is carried out, FIG. 7(b), the assembledstructure is optionally subjected to the anodization process asdescribed earlier, so that both the surface of the Ti wire or meshscreen and the surface of the Ti implant base material are anodized tohave TiO₂ nanotube or nanopore structure for accelerated cell- orbone-growth as indicated in FIGS. 7(c) and (d). The space within each ofthe nanotubes or nanopores can optionally be utilized to storebiological agents such as growth factors, antibiotics, genes, DNAs,therapeutic drugs, metallic or magnetic nanoparticles, etc. to furtheraccelerate the cell or bone growth, or to serve as an implantedtherapeutic medical treatment device.

FIG. 8(a) schematically illustrates a side view of an exemplary hairy orwire-mesh-screen Ti (or alloy), or any biocompatible alloy such asstainless steel, attached onto Ti implant surface for enhancedtoughness, strength, and mechanical locking of bone growth aroundimplant. The spot-welded, induction melting-bonded, DC or AC plasmabonded, e-beam bonded, laser-bonded or braze-bonded Ti wire mesh (singlelayer or multi-layer), having surface TiO₂ nanopore or nanotube array,enables strongly locked-in bone (or cell) growth around wire-shape ormesh-screen shape Ti wires as illustrated in FIG. 8(b), at the same timeallowing accelerated bone or cell-growth due to the presence of surfacenanostructure.

The mechanical bond strength between a growing hard tissue (e.g., bone)and the implant material with protruding structure can further beimproved by introducing a dual-structured, bone-locking Ti implantcontaining recessed cavities, especially those having re-entrant shapedcavities as illustrated in FIG. 9. These cavities have macroscaledimensions with the diameter and depth being in the size range of10-5000 micrometers, or in another aspect, 25-500, and exhibit adesirable bone lock-in structure. These cavities can be formed byphotolithography, shadow-mask lithography, or various othernon-conventional lithography techniques followed by chemical etching.

FIG. 10 illustrates, instead of wire-based protruding structures,exemplary horizontally placed Ti (or alloy) particles (near spherical orshort fiber shape) attached onto Ti implant surface serve as protrudingstructures in this exemplary structure of the invention. The particlescan be in a monolayer configuration as illustrated in FIG. 10 or a threedimensionally-connected configuration as illustrated in FIG. 11. In oneaspect, these particle or fiber aggregate structures attached onto Tiimplant surface can provide enhanced toughness, strength, and mechanicallocking of bone growth around the particle or fiber aggregate.

The particle shape is arbitrarily defined here as a near-sphericalobject with an aspect ratio of less than 2 and having regular orirregular surface. The fiber shape is arbitrarily defined here as anelongated object with an aspect ratio of at least 2 and having regularor irregular surface, straight or bent configuration. The protruding Timicro/macro particles or fibers as shown in FIG. 10 can be attached ontothe Ti implant surface by processing approaches involving inductionmelt-bonding, e-beam melt bonding, laser bonding, spot-welding, ordiffusion-bonding.

The material for these micro/macro particles can be Ti or Ti-base alloys(e.g., Ti—Al—V alloys), other refractory metals (e.g., Zr, Hf, Ta, W andtheir alloys), or stainless steels can also be used for the protrudingstructure as well as for the implant base. The desired diameter of themicro/macro particles or fibers in FIG. 10 and FIG. 11 composing theprotruding structure is in the range of 10-10,000 micrometers, or inanother aspect, in the range of 25-500 micrometers. The desiredthickness of the protruding structure depends on specific applicationsand the average diameter of the particles or fibers involved. In oneaspect, the desired overall thickness of the protruding structure layeris 0.01-10 mm, or in another aspect, 0.05-2 mm.

An exemplary process of fabricating such a structure is described asfollows.

i) The particles or fibers made of Ti or Ti alloys, or otherbiocompatible metals or alloys are prepared or procured, and then areplaced on the top surface of the implant base structure.

ii) One alternative method of fabricating the protruding structure ofFIG. 10 or FIG. 11 comprising particle or fiber aggregate structure is asimple dropping of the Ti or alloy powders or fibers on the Ti implantsurface and sintering at a high temperature in the range of betweenabout 500 to 1000° C., or about 500 to 1100° C., or about 500 to 1200°C., for an exemplary duration of between about 0.1 to 10 hrs, in aninert, vacuum, or reducing gas atmosphere. Ar, He or nitrogen gas can beused for inert gas heat treatment while hydrogen, forming gas (in oneaspect, about 1 to 15% mixture of H₂ in N₂ or Ar gas), or ammonia gascan be used for reducing atmosphere heat treatment. Alternatively,instead of sintering, such a pile of Ti (or Ti alloy) powders or fiberscan be spot welded under compression to attach the particles or fibersamong themselves and to the surface of the Ti implant base. Such a spotwelding is especially desirable from the practical point of view as theprocess is fast and convenient since the complicated step of heattreatment to bond loose powders or fibers is avoided.

iii) After the protruding structure composing of these particle or fiberaggregate layer is attached onto the Ti base implants, the assembly issubjected to the anodization process described earlier, so that both thesurface of the Ti wire or mesh screen and the surface of the Ti implantbase material are anodized to have TiO₂ nanotube or nanopore structurefor accelerated cell- or bone-growth as indicated in FIGS. 6(c) and (d).Other alternative processes such as chemical etching, surface meltevaporation, plasma etching, etc, instead of anodizing, can also beutilized to induce the surface nanostructures of metallic or oxidenanopores, nanotubes, nanowires on the hairy or wire-mesh protrusionstructure.

iv) The assembly is optionally heat treated, e.g., near the temperatureof about 500° C. for 0.1-10 hrs to crystallize the nanotube and obtain adesirable crystal structure such as the anatase phase. The space withineach of the nanotubes or nanopores can optionally be utilized to storebiological agents such as growth factors, antibiotics, genes, DNAs,therapeutic drugs, metallic or magnetic nanoparticles, etc. to furtheraccelerate the cell or bone growth, or to serve as an implantedtherapeutic medical treatment device as described below.

2. Externally and Remotely Controllable Drug-Delivery Systems

The invention also provides externally controllable drug-deliverysystems comprising metallic, ceramic or polymeric materials, and methodsfor operating such systems, an exemplary embodiment being a structure ofmagnetic nanoparticles inserted together with drugs or biological agentsinto nanopores/micropores or into the gaps in nanowire/microwire arraysor nano-ribbon/micro-ribbon arrays, or a structure consisting ofmagnetic wire array or ribbon array.

This aspect of the invention can comprise structures as described, e.g.,in PCT Patent Application no. PCT/US2006/016471, filed on Apr. 28, 2006,Jin et al., e.g., can incorporate a drug delivery system utilizing aTiO₂ nanotube arrays, e.g., as described in Jin et al. This inventionprovides drug delivery systems, either passive or active,remote-actuated, on/off controllable and programmable drug deliverysystems comprising various nanopore structures(non-TiO₂-nanotube-based). FIG. 12 schematically illustrates exemplarydrug delivery systems of the invention. The schematics shown in FIG.12(a) describe passive, diffusion based, slow drug release device. Thedrawing in FIG. 12(b) illustrates an active, remotely and externallycontrollable, drug release device which is an on-command, drug deliverysystem.

In one aspect of the invention, densely spaced Ti particles, fibers orwire arrays are attached onto Ti implant surface by utilizing variousprocessing methods such as diffusion bonding, melt-bonding by rapidheating with laser, electron beam, RF or induction field, DC or Acplasma or spot welding to introduce a microporous structure for use asthe basis of a drug delivery reservoir. The implant base mostly servesas a biocompatible carrier of the microporous structure although some ofits own surface areas also serve to store and release drugs. The drugsor biological agents to be stored and released in a controlled manner inthis invention include pharmaceutical therapeutic drugs such asantibiotics, chemotherapy medicine, anti-stenosis drug, insulin, andbiologically active agents such as DNAs, genes, proteins, hormones,collagens and other growth factors, magnetic nanoparticles,infrared-light-absorbing nanoparticles, etc. Both the particles/fibersand the implant base material can be made of biocompatible metals oralloys such as Ti based alloys, or other refractory based metals andalloys, or stainless steel. The surface of the implant base can haveflat, round or curved surface depending on specific applications.

The surface of each particle or fiber in the microporous aggregate isthen further modified with anodization-induced TiO₂ nanotube ornanopores as illustrated in FIG. 12(a). The process of anodizing Tisurface to prepare TiO₂ nanotube arrays is well established. Seearticles by B. B. Lakshmi, et al., Chemistry of Materials (1997)9:2544-2550, by Miao, et al., Nano Letters (2002) 2(7):717-720, by Gong,et al., Journal of Materials Research (2001) 16(12):3331-3334, by J. M.Macak, et al., Angew. Chem. Int. Ed. (2005) 44:7463-7465, ElectrochimicaActa (2005) 50:3679-3684, and Angew. Chem. Int. Ed. (2005) 44:2100-2102,by A. Ghicov, et al., Electrochemistry Communications (2005) 7:505-509,by S. Oh et al., “Growth of Nano-scale Hydroxyapatite Using ChemicallyTreated Titanium Oxide Nanotubes”, Biomaterials (2005) 26:4938-4943, and“Significantly Accelerated Osteoblast Cell Growth on Aligned TiO₂Nanotubes”, Journal of Biomedical Materials Research (2006) 78A:97-103.

For anodization, in one aspect, fluorine containing chemicals areutilized as an electrolyte, and a voltage of 10-25 volts is applied. Theconcentration of electrolytes has to be carefully chosen, as reported inarticles by Gong, et al., Oh, et al., Macak, et al., and Ghicov, et al.mentioned above. Some exemplary electrolytes and their concentrationsare; 0.5 wt % hydrofluoric acid (HF) in water, 0.5 wt. % ammoniumfluoride (NH₄F) in 1 M ammonium sulphate ((NH₄)₂SO₄), and 1 wt. % NaF in1M Na₂SO₄ solution. Various anodization processing parameters such asthe applied voltage, reaction time, the pH and the temperature of thebath, etc. have to controlled and optimized as well.

The anodized nanopores in these structures, in combination with themicropore base structure, allow a storage of any desired drugs andbiological agents, and their slow, diffusion-based release. Such astructure constitutes the basis of an efficient, biocompatible,time-controlled drug delivery system for pharmaceutical therapeuticdrugs such as antibiotics, chemotherapy medicine, anti-stenosis drug,insulin, and biologically active agents such as DNAs, genes, proteins,hormones, collagens and other growth factors, magnetic nanoparticles,infrared-light-absorbing nanoparticles, etc.

The desired dimension of the particle/fiber aggregate porous structureof FIG. 12(a) is as follows. The average diameter of the particles orfibers can be in the range of about 1-1000 micrometers, or in anotheraspect, 10-250 micrometers. The average diameter of the pores is in therange of about 0.1-100 micrometers. The desired pore volume in theaggregate structure is at least 20%, or in another aspect, at least 50%.The overall thickness of the aggregate structure attached onto theimplant base is in the range of about 10-10,000 micrometers, or inanother aspect, 100-2500 micrometers. The diameter of the nanopores inanodized TiO₂ nanotubes or nanopores is in the range of about 20-500 nm,and the depth of the nanopores (or the height of the nanotubes) is inthe range of about 100-10,000 nm.

The insertion of drugs can be in an aqueous liquid form; insertion intonanopores can be difficult because of the surface tension of the liquidinvolved and the trapped air within the nanopores which tends to blockthe incoming liquid. While a long-time immersion (e.g., 10-100 hrs) ofthe porous aggregate of FIG. 12(a) in a pool of liquid containing thedesired drug is helpful for inserting the drug or biological agentliquid into the nanopores, this invention calls for a unconventionalapproaches to ensure efficient insertion of drugs or biological agentsinto the nanopores.

(a) Use of supercritical CO₂ deposition technique—Supercritical carbondioxide (scCO₂) exhibits a novel hybrid characteristics of liquid-likeand also vapor-like properties. Like a liquid, it can dissolve solutes,e.g., some drugs in the present invention. Like a vapor, it possesseslow viscosity, high diffusivity and negligible surface tension, so itcan deliver chemicals and drugs into nanoscale cavities orhigh-aspect-ratio nanopores. Examples of scCO₂ processing to delivermaterials into small nanopores are described, for example, in articlesby Ye et al., Xiang-Rong Ye, Yuehe Lin, Chongming Wang, Chien M. Wai,Adv. Mater. (2003) 15:316, and Xiang-Rong Ye, Yuehe Lin, Chongming Wang,M. H. Engelhard, Chien M. Wai, J. Mater. Chem. (2004) 14:908.

(b) Use of vacuum or boiling water process—Often the trapped air insidenanopores or micropores can prevent the insertion of drug-containingaqueous solution. In this invention, vacuum (such as 10⁴-10⁶ torr levelvacuum obtained by pumping a chamber using a mechanical pump, diffusionpump, cryopump, or turbo pump) can be used to remove the trapped airprior to letting the drug-containing aqueous solution into the chamberand hence inside the nanopores or micropores of FIG. 12(a) type drugdelivery reservoir material placed in the chamber. Another techniqueutilized in this invention is to place the drug delivery reservoirmaterial inside a drug-containing aqueous solution, and boil thesolution so that the trapped air from the nanopores is removed and thesolution gets inside the nanopores. This technique is of course suitableonly when the drug or biological agents to be inserted does not getdamaged on exposure to the boiling temperature.

(c) Use of pressure injection process—A liquid containing the drug,biological agent or magnetic nanoparticles can be loaded into thenanopores or micropores by using high pressure injection or infiltrationtechnique.

The speed of the drug release is controlled/programmed by design of themicro particle aggregate structure and the TiO₂ nanotube typenanostructure. In this invention, two major materials parameters arecontrolled to optimize the drug release rate.

i) The volume fraction and diameter of the Ti micro particles or fibersin the aggregate, and hence the size, the lengths of the micropores andvolume fraction of the micropores between the microparticles, and

ii) The diameter, spacing and depth of the TiO₂ nanotubes on the surfaceof each of the micro particles or micro fibers, within which the drugsor biological agents are stored. These dimensions dictate the overalldiffusion distance and time required for the released drug to reach thesurface of the particle/fiber aggregate to become available for in-vivobio interactions or chemical reactions.

Referring again to FIG. 12(b), the device illustrates an active,remotely and externally controllable, drug release device operated byapplied magnetic field. In one aspect of the invention, the drugs orbiological agents to be stored and released such as pharmaceuticaltherapeutic drugs (such as antibiotics, chemotherapy medicine,anti-stenosis drug, insulin), and biologically active agents (such asDNAs, genes, proteins, hormones, collagens and other growth factors) areinserted into the micropore or nanopore reservoirs in FIG. 12(b) typestructures together with magnetic nanoparticles. The drugs or biologicalagents can be dissolved in liquid or contained as a colloidal suspensionmixture. Various techniques including soaking in drug-containing liquid,supercritical CO₂ processing, vacuum suction, pressure injection,boiling water processing, etc. can be utilized for the insertion.

In one aspect, the invention provides an on-command, drug deliverysystem. The exemplary device of FIG. 12(b) requires the incorporation ofmagnetic nanoparticles such as biocompatible Fe₃O₄ (magnetite) or Fe₂O₃(maghemite) nanoparticles with an average diameter in the range ofabout, e.g., 2-50 nm, or in another aspect, 5-20 nm. These magneticparticles can be magnetically moved or vibrated utilizing a DC magneticfield (or very low frequency ac field) with time-dependent changingfield directions to allow the movement and release of thedrug-containing solution from the nanopore or micropore reservoir which,in the absence of such magnetic stimulation, tends to be retained withinthe nanopores or micropores by capillary confinement.

An alternative embodiment is that instead of movement of magneticparticles, they can be stationary, but can be selectively heated byexternal ac magnetic field, e.g., at about 100 KHz, which alsoselectively heats up the drug-containing aqueous solution nearby.Magnetic particles have been used for local heating, for example forin-vivo magnetic hyperthermia treatment of cancer; or as described in,e.g., Pankhurst et al., Journal of Physics D: Appl. Phys. (2003) 36:R167-R181; Tartaj, et al., Journal of Physics D: Appl. Phys. (2003)36:R182-R197; PCT/US04/043459, filed on Dec. 23, 2004, Jin, et al.

The heated drug-containing solution in the nanopore or microporereservoir is then diffused out at an accelerated pace. In one aspect ofthe invention, these magnetically remote-controllable drug releasedevice can be basically turned on or turned off at will by switchingon/off of the applied magnetic field. Another advantageouscharacteristics of the inventive magnetic drug release device is aquantitatively control of the amount of the drug released by theduration and the number of repeated cycles of magnetic heatingoperations actuated by externally applied magnetic field.

FIG. 13 schematically illustrates another embodiment of the inventive,remote-controllable, on-command drug delivery system. Here, a mesoporousaggregate material filled with magnetic nanoparticles are utilized asthe reservoir for storage of drugs or biological agents. First,mesoporous aggregate material is attached onto a substrate or supportmaterial which can be selected from biocompatible material such as Ti,noble metal, ceramic, polymer, or any material coated with biocompatiblesurface layer. This is schematically illustrated in FIG. 13(a). Theattachment can be accomplished by, e.g., diffusion bonding,melt-bonding, adhesive bonding, etc. Examples of mesoporous aggregatematerials useful for this embodiment of the invention include mesoporouscarbon, mesoporous silicon, mesoporous metal, mesoporous ceramic, ormesoporous polymer aggregates. Exemplary size of the magneticnanoparticles can in one aspect be in the range of about 5-20 nm. Thetypical desired diameters in the mesoporous aggregate to store the drugsor biological agents together with magnetic nanoparticles, is in therange of about 5-500 nm, or in another aspect, 5-100 nm.

An aqueous solution of drugs or biological agents, together withmagnetic nanoparticles is then infiltrated into the nanopores ormicropores in the mesoporous aggregate using the techniques describedearlier, i.e., supercritical CO₂ infiltration technique, vacuum suction,pressure injection, or boiling water technique. Such mesoporousaggregate containing drugs or biological agents, together with magneticnanoparticles is illustrated in FIG. 13(b). The magnetic particles inthe nanopores or micropores of the mesoporous aggregate can bemagnetically moved or vibrated utilizing a DC magnetic field (or verylow frequency ac field) with time-dependent changing field directions toallow the movement and release of the drug-containing solution from thenanopore or micropore reservoir which, in the absence of such magneticstimulation, tends to be retained within the nanopores or micropores bycapillary confinement.

In one aspect, instead of movement of magnetic particles to release thedrug, the magnetic nanoparticles are heated to initiate the drugrelease. The magnetic particles can be selectively heated by external acmagnetic field, e.g., at about 100 KHz, which also selectively heats upthe drug-containing aqueous solution nearby. The heated drug-containingsolution in the nanopore or micropore reservoir of the mesoporousaggregate is then diffused out at an accelerated pace. In one aspect,these magnetically remote-controllable drug release device can bebasically turned on or turned off at will by switching on/off of theapplied magnetic field. Alternative characteristics of the inventivecomprise a magnetic drug release device having the ability to release aquantitatively controlled amount of the drug by the duration and thenumber of repeated magnetic heating cycles applied to the in-vivo drugreservoir using external applied field.

FIG. 14 schematically illustrates an exemplary drug delivery system withnanowire, microwire or micro-ribbon array that holds a drug orbiological agent and releases it by remotely activated magnetic field.The forest can exhibit either vertically aligned structure or somewhattangled, non-vertical-aligned structure. Two different types of drugrelease actuation are described, i.e., by the movement of magnetic wiresor ribbons themselves (FIGS. 14(a) and (b)), or by the movement ofmagnetic nanoparticles placed within the drug-containing liquid storedin the wire or ribbon forest (FIG. 14(c)). The drugs or biologicalagents to be stored and released such as pharmaceutical therapeuticdrugs (such as antibiotics, chemotherapy medicine, anti-stenosis drug,insulin), and biologically active agents (such as DNAs, genes, proteins,hormones, collagens and other growth factors) are inserted into theforest of nanowire, microwire or micro-ribbon arrays by varioustechniques including soaking, supercritical CO₂ processing, vacuumsuction, pressure injection, boiling water processing, etc.

In FIG. 14(a), a forest of magnetically actuate-able nanowire (such ascarbon nanotube forest coated with magnetic material), magneticnanowire, magnetic micro-wire, or magnetic micro-ribbon is prepared on abiocompatible substrate. The drug or biological agent iscapillary-trapped in the forest of the in-vivo drug storage device ofthe type illustrated in FIG. 14, which is released by magneticallyinduced movement of magnetic wires or ribbons when they are actuated tomove by remotely applied magnetic field of regular, sequential orgradient in nature.

The material for the magnetic nanowires, micro-wires or micro-ribbonshas to ferromagnetic such that a sufficiently strong response to appliedmagnetic field is exhibited to squeeze out the stored, drug orbiological agent in the forest structure, as illustrated in FIG. 14(b).Exemplary ferromagnetic materials include Ni—Fe permalloys. Fe-base,Ni-base, or Co-base soft magnetic amorphous alloys (e.g., Metglas typematerials). They should also be biocompatible or coated withbiocompatible material such as Ti, Au, Pd, Pt, stainless steel orbio-inert polymers. The desired diameter or thickness of the nanowires,micro-wires or micro-ribbons is in the range of about 0.005-250micrometers, or in another aspect, 0.01-50 micrometers. An exemplary gapbetween adjacent wires or ribbons is in the range of about 0.01-50micrometers.

In FIG. 14(c), the nanowires, microwires or micro-ribbons that form thedrug-containing forest are non-magnetic. Carbon nanotubes or othermetallic or polymer nanowires, microwires or micro-ribbons can beutilized as the forest material. In order to induce the magnetic fieldinduced drug release, magnetic nanoparticles such as Fe₃O₄ nanoparticlesare added to the drug solution. An application of high frequency acmagnetic field (e.g., in the range 10 KHz-10 MHz, or in another aspect,near 100 KHz) make the magnetic nanoparticles and the drug-containingliquid nearby to get preferentially heated so that a diffusion baseddrug release is accelerated.

Yet another embodiment configuration of drug release device usingmagnetic remote control is illustrated in FIG. 15. This approach isbased on the formation of directionally etched porous material which isthen filled a liquid containing drug or biological agent, together withmagnetic nanoparticles. The directional pores do not have to beprecisely vertical from the drug release point of view. They can betilted or irregular shaped. As long as the pores are generally alignedand continuously open to the bottom of the pore, they will serve thepurpose. The process of fabricating such a drug delivery system isillustrated by FIG. 15(a)-(d).

First, a suitable base material that can be directionally etched isselected as illustrated in FIG. 15(a). Example base materials to etchand form directional nano or micro-pores include Al, Si, ceramics,various metals including Ti or Ti-base alloys (e.g., Ti—Al—V alloys),other refractory metals (e.g., Zr, Hf, Ta, W and their alloys), orstainless steels, metal-ceramic composites, or polymers. It can be asingle phase material. Alternatively, two-phase or composite materialswith vertically textured, two-phase alloys or ceramic, or diblockcopolymers can advantageously be utilized for ease of selective anddirectional etching for vertically aligned pore formation.

In FIG. 15(b), desirably directionally porous structure is formed byvarious types of chemical, physical or thermal etching. Verticallyporous ceramic (e.g., anodized aluminum oxide or Ti oxide), porous Si,porous metal/alloy, porous polymer, prepared by chemical orelectrochemical etching, thermal or plasma etching utilizingdifferential melting point or differential vapor pressure (evaporationrate) of component metallic phases, differential sputter etch rate orion etch rate (crystal orientation dependent or two phase'scomposition-dependent), or post-thermal-process chemical etching such ason melt textured (directional solidified) structure by induction, laseror e-beam melting, or sputter/resputter process.

In one aspect, the surface of these pores needs to be biocompatible foran in vivo drug delivery system. If the base material itself is notbiocompatible, they can be coated with a layer of biocompatible materialsuch as Ti, Au, Pd, Pt, stainless steel or bio-inert polymers bicoatedif needed.

FIG. 15(c) illustrates one aspect of the invention where an optionalpartial capping that reduces the pore entrance size. Such a partialcapping helps to prevent the escaping of magnetic particles to retainthe remote control capability, and also to minimize any side effect onrelease of loose magnetic particles in the in-vivo system. In oneaspect, the partial capping of drug-releasing pores can be accomplishedby oblique incidence sputtering or evaporation, quick electroplating,quick electroless plating, or quick dipping in adhesives.

In FIG. 15(d) illustrates one aspect of the invention where variousdrugs or biological agents are be stored and released, e.g., in oneaspect pharmaceutical therapeutic drugs (such as antibiotics,chemotherapy medicine, anti-stenosis drug, insulin) and/or biologicallyactive agents (such as DNAs, genes, proteins, hormones, collagens andother growth factors) are inserted into the micropore or nanoporereservoirs into the FIG. 15(b) or FIG. 15(c) type structures togetherwith magnetic nanoparticles. The drugs or biological agents can bedissolved in liquid or contained as a colloidal suspension mixture.Various techniques including soaking in drug-containing liquid,supercritical CO₂ processing, vacuum suction, pressure injection,boiling water processing, etc. can be utilized for the insertion.

In one aspect, the drug delivery system of the type described in FIG.15(c) or FIG. 15(d) is then in-vivo implanted, and remote magnetic fieldactuated for on-command release of drugs or biologically active agentsby magnetic field on/off operation or quantitative dose control usingselected magnetic field intensity/frequency/repetition/duration.

FIG. 16 schematically illustrates an exemplary magnetic remotecontrollable drug delivery system based on porous structures made byevaporated or sputtered thin or thick films. In one aspect, both thefilm and the substrate has to be biocompatible for in-vivo drug deliveryuse. Ti or TiO₂ based thin films (or other refractory metal/alloys andtheir oxides, noble metals/alloys, stainless steels) can be used as thethin film or substrate material. The porous films in FIG. 16(a) can befabricated either as an as-deposited film or as a post-deposition-etchedfilm with one of the phases in a multi-phase microstructure etched away.

In one aspect, a proper selection of sputtering pressure and temperaturecan introduce porous or rough microstructure in deposited thin films.The self shadowing effect of the obliquely deposited thin film material,e.g., by evaporation can be used to form highly porous or rough films.The sputtering deposition can be carried out by DC, pulse DC, RFsputtering, or ion beam deposition methods. The evaporation can be doneby thermal or electron beam evaporation process. Depending on thedeposition conditions, a smooth continuous film, rough topology film, orhighly porous structure can be obtained. See e.g., J. A. Thornton, J.Vac. Sci. Technol. (1986) A4(6):3059, L. J. Meng et al., “Investigationsof titanium oxide films deposited by dc active magnetron sputtering indifferent sputtering pressures,” Thin Solid Films (1993) 226:22, by K.Robbie et al., “Fabrication of thin films with highly porousmicrostructure,” J. Vacuum Science & Technology (1995) 13(3):1032, andJ. Rodriguez et al., “Reactively sputter deposited titanium oxidecoatings with parallel Penniform microstructure,” Adv. Mater. (2000)12(5):341.

In one aspect, once such a porous film structure of formed, asillustrated in FIG. 16(a), biological agents such as growth factors,collagens, hormones, DNAs, etc. can be inserted into the porous filmstructure as illustrated in FIG. 16(b) for enhanced in-vivo or in-vitrobio activities such as accelerated cell growth, enhanced hormone oralbumin secretion, increased protein synthesis from the cells involved,etc.

FIG. 16(c) illustrates one embodiment where a drug in liquid (eitherdissolved or as a colloidal mixture with the carrying liquid such aswater or body fluid), together with magnetic nanoparticles, is insertedinto the nano or micro pores in the thin film. Various techniquesincluding soaking in drug-containing liquid, supercritical CO₂processing, vacuum suction, pressure injection, boiling waterprocessing, etc. can be utilized for the insertion. The drug deliverysystem of the type described in FIG. 16(c) is then in-vivo implanted asillustrated in FIG. 17, and remote magnetic field actuated foron-command release of drugs or biologically active agents by magneticfield on/off operation or quantitative dose control using selectedmagnetic field intensity, frequency, repetition cycles, and duration ofthe magnetic field.

3. Elastically Compliant Implant Material for Bone Growth

Alternative embodiments of the invention comprise a unique configurationof elastically compliant implant material for, e.g., bone growth, whichprovides subdivided, more reliable, and stress-accommodating implantfibers more or less vertically arranged in a spring configuration, andwhich also provides a strong mechanical reinforcement of the grown bonevia bone-metal wire composite formation. In one aspect, Ti based metalor alloy or any biocompatible alloy such as stainless steel can beutilized, e.g., minimal separation failures at the implant-hard tissueinterface, which also provides strength and toughness reinforcement ofthe grown bone via bone-metal wire composite formation.

Exemplary structures of such elastically compliant implant material forbone growth is illustrated in FIG. 18. On the surface of Ti or Ti alloyimplant (flat, round or curved surface), an array of verticallyspring-configured Ti wires (FIG. 18(a)) or spring-configured Ti meshscreens (FIG. 18(b)) or any other spring-like structure of Ti wires orribbons are bonded, for example, by using spot welding procedure asillustrated in FIG. 18(c) and FIG. 18(d). Optional spacer/protector mayalso be added onto the surface of the Ti implant base in order toprotect the Ti spring members during abrasive insertion of Ti implants(e.g., screw-like implants into bones or teeth).

Compliant, springy, or bent Ti (wire, ribbon, mesh screen of pure metalor alloy) in macroscale (e.g., the diameter/thickness of the wire/ribbonis more than about 1-10 micrometers) can be made by employingpre-multiple-bent Ti wires or ribbons cut to desired length. In the caseof micro or nanowires of Ti, these can be made by oblique incidentevaporation or sputtering. These spring-configured Ti springs in wire ormesh-screen form can be attached onto the Ti implant surface bydiffusion bonding, brazing, induction-melt-bonding, e-beam melt-bonding,laser-melt-bonding, spot welding, etc.

The surface of the Ti wires or ribbons as well as the surface of the Tiimplant base can be optionally anodized to form cell- or bone-growthaccelerating TiO₂ nanotubes or nanopores, as made by using theanodization process described earlier.

Instead of using Ti based metals or alloys (e.g., Ti—Al—V alloys), orany biocompatible alloy selected from refractory metals (e.g., Zr, Hf,Ta, W and their alloys), or stainless steels can be employed as thespring material or the implant base material. If the spring materialselected is not biocompatible, its surface can be coated with abiocompatible or noble metal, alloy or polymer.

FIGS. 19(a) and (b) describe the alternative embodiments comprising bonegrowth steps around compliant Ti spring material, the spring nature ofthe wires or ribbons accommodate any stress applied (shear, tensile orcompressive stresses) during the bone growth, thus preventing thefracture or delamination of the newly growing bone. In one aspect, oncethe bone growth is completed filling the gap between the implant andexisting bone, the presence of Ti wires, ribbons, mesh-screens withinthe grown bone serves as reinforcement as in a reinforced concrete, thusminimizing failures of the attached bone.

4. Non-Metallic or Non-Ti Based Substrates with their Surfaces Convertedto TiO₂-Type Nanotubes or Nanopores

Additional exemplary embodiments comprise utilizing non-metallic ornon-Ti based substrates and converting their surfaces into TiO₂ typenanotubes or nanopore so as to exhibit desirable cell or bone growthaccelerating characteristics. Examples of non-Ti type, nanoporous ormicroporous materials include anodized Al₂O₃ membrane, porous Si, porouspolymer, and porous metals and alloys in general, as illustrated in FIG.20(a).

In one aspect, two methods of accomplishing such a biocompatiblemodified structure are disclosed. One method is to coat the surface ofthe materials (which in one embodiment have been processed to havenanopores before the coating) (in alternative aspects the materials cancomprise anodized alumina, porous silicon, diblock-copolymer-basedporous polymer, or any combination thereof) with a thin andbiocompatible Ti or TiO₂ type layer, which in alternative embodimentscan be between about 1 to 50 nm thick layer, by sputtering, evaporation,chemical vapor deposition, plasma spray, thermal spray, etc. Anexemplary process is illustrated in FIG. 20. A thin biocompatiblesurface coating of Ti, TiO₂ or other related biocompatible metals andalloys are deposited by physical vapor deposition (such as sputtering orevaporation) or chemical vapor deposition, as illustrated in FIG. 20(b).The accelerated cell growth on such a nanostructure with biocompatiblecoating is shown in FIG. 20(c).

Another exemplary method of the invention is to apply a thick layercoating of Ti or related metals, including a Ti, Zr, Hf, Nb, Ta, Moand/or W metal; a Ti, Zr, Hf, Nb, Ta, Mo and/or W alloy; and/or, a Ti,Zr, Hf, Nb, Ta, Mo and/or W oxide or nitride, and/or stainless steel orceramic, or any combination thereof; which in alternative embodimentsare applied at 100-2000 nm thicknesses; and in alternative aspects areapplied on a generally smooth, but not nanoporous and at mostmacro-porous, Si, Si oxide, carbon, diamond, noble metals (such as Au,Ag, Pt and their alloys), polymer or plastic materials, or compositemetals, ceramics or polymers, and then anodizing the thick Ti or relatedmetals and converting at least a portion of the Ti or related metalsurface into TiO₂ type nanotubes or TiO₂-surfaced nanopores.

The products of manufacture of the invention can have either the thin orthick layer coating, or combination thereof, and can be used for anyvariety of applications as described herein, including for in vitrotesting of drugs, chemicals or toxins, or as in vivo implants, use inmaking and using artificial tissues and organs—which includes cell, boneand tooth growth, and as drug delivery devices.

The drawings in FIG. 21 schematically illustrate this exemplary processof creating a TiO₂ nanotube or nanopore surface structure on non-Ti typesurfaces by thick Ti film deposition followed by anodization. Theexemplary process used in FIG. 21 also can comprise a formation ofre-entrant loop shape cavity, which might be useful to, e.g., furtherlock-in growing bones, teeth or other tissues.

Shown in FIG. 22 is the nature of cell- or bone-growth-acceleratingcoating of TiO₂ nanotubes or nanopores by anodization of thick-film Ticoating on an exemplary pre-patterned, non-Ti type substrate (ceramics,polymers, plastics, Si, Au, Pt, Al, etc.), and resultant cell or bonegrowth with a mechanically more reliable lock-in structure.

FIG. 23 schematically illustrates various potential in vivo or ex-vivobio implant applications of the inventive, biomaterials capable ofaccelerated cell/bone/teeth growth, strongly locked-in bone or cellgrowth, or functional drug delivery and therapeutics. Various inventivenanopore or nanotube structures described in relation to FIGS. 1-22 canbe utilized for these biomedical device applications. Examples showninclude orthopedic and dental implants, cell or organ implants, drugdelivery devices such as controlled release of insulin by magneticactuation, artificial liver devices, drug-protected stents (e.g., toprevent/minimize restenosis) or other tubules inserted into bloodvessels and in various other body parts, and therapeutic devices such asmagnetic field induced local heating for cancer treatment. Cell growthinhibiting drugs can also be inserted in the inventive nanopores ornanotubes of other implants (e.g., drug delivery modules) toprevent/minimize scar tissue formation.

The various large-surface-area biomaterials described in relation toFIGS. 1-22 can be useful for ex-vivo accelerated cell growth devices.Rapid production of healthy cells including liver cells, bone cells,kidney cells, blood vessel cells, skin cells, periodontal cells, andstem cells can be realized. A device application of such an ex-vivoaccelerated cell growth is shown in FIG. 24, which uses an example of anarray of protruding, large-surface-area, hairy, mesh-screen orparticulate shaped Ti members bonded onto Ti substrate and processed tohave TiO₂ nanotube or nanopore surface, for applications such as cellsupply, rapid cell identification, harvesting of cell components,secreted proteins, albumin and other bio components generated bycultured cells. Other types of large-surface-area biomaterials such asillustrated in FIGS. 10-22 can also be utilized for such rapid cellgrowth.

FIG. 25 schematically illustrates an exemplary accelerated liver cellgrowth device comprising an array of protruding, large-surface-area,hairy, mesh-screen, particulate, or particulate-aggregate shaped Timembers bonded onto Ti implant surface and processed (e.g., byanodization) to exhibit TiO₂ nanotube or nanopore surface, forapplications such as rapid toxicity testing of drugs or chemicals. Manynew types of therapeutic or analytical drugs are explored and developedevery year by pharmaceutical companies and other biotech R&D institutes.If a drug is toxic to human or animal body under in vivo situation, theliver is one of the first organs to sense it and try to isolate thetoxic materials. A bio-chip containing an array of healthy,three-dimensionally cultured liver cells, e.g., 10.times.10,100.times.100 or 1000.times. 1000 sensing elements can thus allowsimultaneous evaluation of many drugs for much accelerated screening anddevelopment of biologically acceptable drugs. Likewise, many chemicals,polymers, injection fluids, and composites that may be useful for invivo applications can be rapidly tested for toxicity using the inventivedevice of FIG. 25. The accelerated, healthy liver cell growth madepossible with various large-surface-area biomaterials described inrelation to FIGS. 1-22 can also be utilized as the basis of artificialliver devices for patients waiting for transplant or as a temporary aidto liver function after transplant. Other functional organs applicationssuch as artificial kidney can also be considered.

FIG. 26 schematically illustrates exemplary embodiments of the inventivebio-chip test apparatus useful for drug toxicity, chemical toxicity, orcell identification testing, with the apparatus comprising an array ofthe inventive, protruding, large-surface-area biomaterials ornanoporous, microporous materials, with the analytes detected by (a)optical means (such as microscopy using visible, infrared or UV light),laser, (b) chemical or biological analysis (such as using assays forchemical or biological reactions in combination with various analyticaltools), or (c) magnetic sensor technique (such as magnetoresistanceprobes based on GMR sensors, TMR sensors or SQUID magnetometers) whichmeasure the change in the position or binding of magnetic nanoparticlesvia the change in the magnitude of detected magnetism.

5. Biocompatible Materials Configured in Loose Particles, LooseShort-Fibers, or Loose Flakes

The invention also provides biocompatible materials configured in looseparticles, loose short-fibers, or loose flakes, with each of theseparticles or short fibers having their surfaces covered with cell- orbone-growth-accelerating nanotube or nanopore array structure. Theseloose powder configurations allow convenient in-vivo or in-vitroimplementations of, e.g., a paste type or bone cement type applications.Because of the presence of the very-large-surface-area, biocompatible,cell-activity-accelerating structure such as titanium oxide nanotubes ornanopores on the surface of such particles, short fibers, or flakes,significantly accelerated and viable cell growth and bone growth occurs.In one aspect, the compositions of the invention (e.g., products ofmanufacture, such as arrays, drug delivery devices) comprise structuresas described, e.g., in PCT/US2006/016471, filed on Apr. 28, 2006, Jin etal.; Oh et al., “Growth of Nano-scale Hydroxyapatite Using ChemicallyTreated Titanium Oxide Nanotubes”, Biomaterials (2005) 26:4938-4943;“Significantly Accelerated Osteoblast Cell Growth on Aligned TiO₂Nanotubes,” J. Biomedical Materials Research (2006) 78A:97-103.

One embodiment comprises a loose particle, short-fiber or flake shapedbiomaterial configuration that allows pharmaceutical drugs, growthfactors and other biological agents to be added and stored within thenanotubes or nanopores for multifunctional advantages, and inalternative aspects allows slow, diffusional, time-dependent release foreven further accelerated growth of healthy cells.

One embodiment comprises an optically transparent or translucentcell-culturing substrate with nano imprint patterned nanostructure.These devices of the invention provide the optical transparency neededby a cell culture substrate, and this embodiment allows a microscopicexamination of the cell behavior using inverted microscope withtransmitted light illumination. In one aspect, a surface of such ananostructure is coated with an optically transparent or translucent,very thin film of, in alternative embodiments: Ti or Ti-base alloys(e.g., Ti—Al—V alloys), other refractory metals (e.g., Zr, Nb, Hf, Ta, Wand their alloys), or TiO₂, Nb₂O₅, ZrO₂, HfO₂, Ta₂O₅, W₂O₃ or mixedalloy oxide. In one aspect, the desired thickness of such Ti or TiO₂related coating is about 1 to 50 nm, or about 1 to 20 nm, or inalternative embodiments: at least 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46,47, 48, 49 or 50 or more nm.

One embodiment comprises an elastically compliant nanostructuresubstrate coated with Ti, TiO₂ or related metal and metal oxide films,including a Ti, Zr, Hf, Nb, Ta, Mo and/or W metal material, a Ti, Zr,Hf, Nb, Ta, Mo and/or W alloy, a Ti, Zr, Hf, Nb, Ta, Mo and/or W oxideor nitride, and/or stainless steel or ceramic. These devices of theinvention help ameliorate the stress or strain that thegrowing/propagating cells experience, and thus have a positive effect onthe cell growth behavior. By providing elastically soft substrate whichis made even more flexible by virtue of added surface nanostructure, afurther enhanced cell growth is obtained using these exemplary devicesof the invention.

In one embodiment, drugs or biological agents are stored inside theloose or fixed structures on the surfaces of devices of this invention,and/or in the pores of TiO₂ type nanotubes, nanowires and/or nanoporeson the surface of the particles, short fibers, or flakes. In oneembodiment, the slowly released compounds include pharmaceuticaltherapeutic drugs (such as antibiotics, chemotherapy medicine,anti-stenosis drug, insulin), and/or biologically active agents (such asDNAs, genes, proteins, hormones, collagens and other growth factors suchas BMP (bone morphological protein)). The drugs or biological agents canbe dissolved in liquid or contained as a colloidal suspension mixture.Various techniques including soaking in drug-containing liquid,supercritical CO₂ processing, vacuum suction, pressure injection,boiling water processing, etc. can be utilized for the insertion.

Another embodiment comprises use of magnetic nanoparticles inserted intothe nanopore reservoirs together with drugs or biological agents so thata remote, magnetic-field-controlled, on-demand delivery of drugs orbiological agents can be accomplished with enhanced kinetics of deliveryin vivo, based on a mechanism of magnetic particle movement in the drug-or biological-agent-containing liquid when a gradient ororientation-changing magnetic field is applied, or on a mechanism ofmagnetic hyperthermia type, preferential heating of magneticnanoparticles when a high frequency ac field actuation, e.g., at 100KHz, is applied.

One exemplary step to make a cell-growth-accelerating cement comprisingTi is to prepare the Ti particles, short fibers or flakes, with adesired size of average diameter (or thickness in the case of flakes) inthe range of approximately 0.2 to 2000 micrometers, or in anotheraspect, or in the range of about 2 to 500 micrometers. They can besynthesized by using a number of different methods such as; i)atomization, ii) plasma spray, iii) chemical precipitation anddecomposition or heating, iv) evaporated or sputtered film depositionand scraping off the substrate or chamber wall where the coating iddeposited. These loose particles, loose short-fibers, or loose flakesare then subjected to anodization treatment to convert the surface intoTiO₂ nanotube, nanowire and/or array structure. An exemplary anodizationprocess is to use 5 wt % HF in water, at an applied voltage of about 20V, and anodization duration of 1-100 minutes. After the anodization, theloose particles, loose short-fibers, or loose flakes with a TiO₂nanotube, nanowires and/or array surface can optionally be heated toabout 300-800° C. for phase changes from amorphous to crystallized phasesuch as the anatase phase.

The drawing of FIG. 27 describes an exemplary method of creating loose,TiO₂-nanofiber-coated or TiO₂ nanotube-coated Ti powder (spherical,elongated, random or short-wire shape particles) by rotating or movingthe electrode (alternatively, the bottom electrode can stay still butthe powders are now agitated instead, and made to move around tooccasionally touch the electrode). The surface of the Ti particles canbe anodized when they are in electrical contact with the rotatingelectrode. Repeated tumbling of the particles can allow additionalsurface regions of the Ti particle to be anodized.

FIG. 28 schematically illustrates an alternative technique of creatingTiO₂ nanotube-coated or TiO₂-nanofiber-coated Ti powder of micro-sizedor macro-sized (spherical, elongated, random or short-wire shape orflake-shape particles. The starting Ti particles (which may have somenative oxidized TiO₂ oxide or suboxide surface can be spherical orrandom in shape, and have desirable, average particle diameter in therange of about 0.2 to 2000 micrometers, or in another aspect, about 2 to500 micrometers. The Ti powder is first subjected to a NaOH chemicaltreatment, e.g., by soaking in about 0.01-5 Normal (in one aspect, about0.5-2 N) NaOH solution, for about 20-120° C./1 sec-1 hr, e.g., for about5 sec-500 sec, to form a surface intermediate phase of sodium titanatenanotubes or nanofibers. The sodium titanate can be expressed with anexemplary formula of Na₂Ti₂O₅H₂O type hydroxide or Na₂Ti₃O₇, but othervariations are also possible.

These sodium titanate nanotubes or nanofibers are then converted intoTiO₂ nanofibers or nanotubes by hydrothermal treatment, e.g., by heatingin boiling water at about 20-120° C. for about 1 sec-1 hr, or about 5sec-500 sec. The water takes away sodium from the sodium titanate andleaves only titanium oxide material behind.

After the anodization, the loose particles, loose short-fibers, or looseflakes with a TiO₂ nanotube or nanofiber array surface can optionally beheated to about 300-800° C. for phase changes from amorphous tocrystallized phase such as the anatase phase. The heating rate to thecrystallization temperature is important as too fast heating tends todestroy the crystal shape and introduces an undesirable internal stressin the material. A heating rate of slower than 10° C./min, or in anotheraspect, slower than about 2° C./min is desired.

An alternative exemplary method of synthesizing the desiredTiO₂-nanotube-covered loose particles or loose short-fibers, or looseflakes for biocompatible, cell- or bone-growth accelerating cement typeapplications, is to utilize a novel processing method, e.g., asdescribed in FIG. 29. The drawing schematically illustrates an exemplaryprocess of utilizing an extended anodization to create thinner andgrind-able TiO₂ wire or ribbon. Thin Ti wires (with a spherical, oval,irregular or random cross-sectional shape) or ribbons, with a diameteror thickness in the range of about 0.2-2000 micrometers, or in anotheraspect, about 2-500 micrometers, average size, are subjected to anexemplary anodization process, e.g., 5 wt % HF in water, at about 20 Vapplied voltage for a duration of 1-100 minutes.

The anodization process is a combination of etching away surfacematerial and an addition of oxidized layer on the material surface. Ause of extended anodization time creates a sufficient TiO₂ penetrationinto the thickness of the Ti wires or ribbons to make the wires orribbons less ductile. In order to make a good use of such inducedbrittleness, a sufficient thickness of the oxide layer relative to theremaining, ductile, metallic core materials is essential. The desiredvolume of the TiO₂ surface layer formed is at least 50% of the overalldiameter or thickness of the Ti wire or ribbon, or in another aspect, atleast 90%. The predominantly TiO₂ wires or ribbons are then subjected tomechanical grinding, pulverization, or ultrasonic sonication in a liquidso as to produce powders, short fibers, or flakes, each segment having acell-growth-accelerating TiO₂ nanotube, nanofiber or nanopore surfacestructure. The desired diameter of the p powders, short fibers, orflakes can in one aspect be in the range of about 0.1-100 micrometers,and the desired length in the case of fibers or flakes can in one aspectbe in the range of about 10-5,000 micrometers.

An optional crystallization heat treatment can be given before or afterthe grinding operation. The crystallization heat treatment by heating toabout 300-800° C. introduces a phase changes from amorphous tocrystallized phase such as the anatase phase.

Such TiO₂-nanotube-coated loose powders, short fibers, or flakes of Tican be useful as a component of accelerated-bone-growth cement. Asillustrated in FIG. 30, the TiO₂-nanotube-coated powders, short fibers,or flakes of Ti, as well as small fragmented pieces of mesh screen ofTiO₂ (with optional Ti core), can be applied near the in-vivo locationwhere accelerated bone growth is desi TiO₂ can also be mixed withbone-growth nutrient, hydroxyapatite, natural bone powder,bio-degradable polymer, bio-compatible or bio-inert bone cement,bio-active glass, or optionally with biological agents of growth factorssuch as BMP (bone morphogenic protein) or collagen, or other biologicalagents such as antibiotics, therapeutic drugs, DNAs, genes, hormones,etc. Magnetic nanoparticles may also be added in the bone cementcomprising powders, short fibers, or flakes, or fragmented mesh screensof TiO₂ if a remote, magnetically actuated release of therapeutic drugsor biological agents is desired.

Shown in FIG. 31 is an exemplary dental use of a cement comprisingTiO₂-nanotube-coated powders, short fibers, or flakes, or fragmentedmesh screens of TiO₂ (with optional Ti core) for accelerated dental bonegrowth. The formation of regenerated dental bone occurs in anaccelerated manner on these loose templates of TiO₂ having nanotube ornanopore surface. For further accelerated kinetics of healthy dentalbone growth, the powders, short fibers, or flakes, or fragmented piecesof mesh screen of TiO₂ can also be mixed with bone-growth nutrients,hydroxyapatite, natural bone powder, bio-degradable polymer,bio-compatible or bio-inert bone cement, bio-active glass, or optionallywith biological agents of growth factors such as BMP (bone morphogenicprotein) or collagen, or other biological agents such as antibiotics,therapeutic drugs, DNAs, genes, hormones, etc. Magnetic nanoparticlesmay also be added in the dental bone cement if a remote, magneticallyactuated release of therapeutic drugs or biological agents is desired.

FIG. 32 schematically illustrates an exemplary periodontal use of thebone cement comprising loose TiO₂-nanotube-coated powders, flakes, shortfibers, or fragmented mesh screen of TiO₂ (with optional Ti core) toaccelerate the periodontal tissue growth and cure. For furtheraccelerated kinetics of healthy growth of periodontal tissues, thepowders, short fibers, or flakes, or fragmented pieces of mesh screen ofTiO₂ can also be mixed with optional growth factors, nutrients,antibiotics, hormones, genes, hydroxyapatite, bio-degradable polymer,bio-compatible or bio-inert bone cement, etc.

This invention provides biomaterials comprising large-surface-areatitanium oxide nanotubes, etc., alternative metals and alloys,fabrication methods, device application methods, and biomedical in-vivoor in-vitro applications for strongly adhered, and kineticallyaccelerated bone growth, periodontal cell growth, organ cell growth(liver, kidney, etc.), drug toxicity testing, cell detection, artificialorgans, etc.

6. Optically Transparent or Translucent Cell-Culturing Substrate withNano Imprint Patterned Nanostructure

The invention provides optically transparent or translucentcell-culturing substrates with nano patterned surface nanostructures,for example, by nano-imprinting with stamps or nanoscale etching. Theinvention provides structures with the optical transparency needed by acell culture, and this embodiment allows a microscopic examination ofthe cell behavior using inverted microscope with transmitted lightillumination. In one aspect, a transparent cell culture substrate isnano-patterned or nano-etched first, the surface of which is then coatedwith an optically transparent or translucent, very thin film of Ti orTi-base alloys (e.g., Ti—Al—V alloys), other refractory metals (e.g.,Zr, Nb, Hf, Ta, W and their alloys with Ti, among themselves or withother alloying elements), or oxides such as TiO₂, Nb₂O₅, ZrO₂, HfO₂,Ta₂O₅, W₂O₃ or mixed alloy oxides, or nitrides. In one aspect, thedesired thicknesses (or thinnesses) of the inorganic coatings (e.g., Tior TiO₂ related alloys, oxides, etc) can be in a range of about 1 to 50nm, or preferably in the range of about 1 to 20 nm.

In one aspect, the devices of the invention have optically transparentor translucent films; a metallic coating is generally opaque unless itis made “very thin” (which, in alternative embodiments, can be in therange from about 1 to 100 nm, 1 to 50 nm, or about 1 to 20 nm, or inalternative embodiments: at least 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46,47, 48, 49 or 50 or more nm). In one aspect, the optically transparentor translucent cell-culturing substrate with nano patterned surfacenanostructure comprise one or more of these selected inorganic coatingmaterials (e.g., Ti or Ti-base alloys, e.g., Ti—Al—V alloys, otherrefractory metals, e.g., Zr, Nb, Hf, Ta, W and their alloys with Ti,among themselves or with other alloying elements, or oxides such asTiO₂, Nb₂O₅, ZrO₂, HfO₂, Ta₂O₅, W₂O₃ or mixed alloy oxides, ornitrides); this can allow biocompatible and cell-culture-enhancingsubstrate which allows microscopic examination with transmitted light.

The transparent substrate material to be surface nano-patterned andcoated with a Ti, Ti oxide, Ti nitride or related inorganic film can beselected from transparent thermosetting polymer, transparentthermoplastic polymer, transparent UV-light-curable polymer, ortransparent glass, and equivalent compounds. Exemplary thermosettingpolymers that can be used in the manufacture of compositions of thisinvention include polydimethylsiloxane (PDMS), polymethyl methacrylate(PMMA), melamine, bakelite, or epoxy resins, and equivalent compounds.Exemplary thermoplastic polymers that can be used in the manufacture ofcompositions of this invention include polyethene, polypropene,polystyrene, or poly vinyl chloride, and equivalent compounds. ExemplaryUV-light-curable polymers that can be used in the manufacture ofcompositions of this invention include polydimethylsiloxane (PDMS) orpolymethyl methacrylate (PMMA), and equivalent polymers.

In alternative embodiments, the nano-patterned, see-through cell culturesubstrate with nano-patterned surface structure and coated withbiocompatible, cell-culture-enhancing inorganic thin film, provides anoptical transparency is at least 10%, 20%, 25%, 30%, 35%, 40%, or 50% ormore, or alternatively at least about 40% of the light is sent through(passes through) the substrate.

Shown in FIG. 33 is an example method of fabricating such transparent ortranslucent cell-culturing substrate with nano imprinted patternednanostructure. In this example, a nano or micro stamp made of silicon,metal or ceramic, e.g., fabricated by photolithography or electron-beamlithography process is utilized to impress into a soft matrix asillustrated in FIG. 33(a). Either an uncured thermosetting polymer,heated and softened thermoplastic polymer, uncured glass precursor, orheat-softened glass can be used as the substrate material to beimprinted. After imprinting (FIG. 33(b) and releasing FIG. 33(c)), thenano patterned substrate is coated by a very thin layer of Ti or Ti-basealloys, other refractory metals, oxides or nitrides as illustrated inFIG. 33(d). The coating of these inorganic material layer(s) on thesurface-nano-patterned and transparent substrate (e.g., plastic,elastomeric polymer or glass) can be done by known methods such assputtering, evaporation, atomic layer deposition, chemical vapordeposition, electroless plating or electroplating and other physical orchemical thin film deposition techniques. A good adhesion of the thinfilm coating on the transparent substrate surface is important. Ti,Ti-oxide or Ti-nitride coating and related refractory metals andcompounds generally provide good adhesion onto substrate surface. Thecoating can be multi-laminar (multilayered) with one or a mixture ofthese compounds.

The nanopattern introduced by imprinting into the plastic, polymer orglass cell-culture substrate can be either periodic or random in itsorder aspect. While a periodic structure can be useful, sometimes arandom structure is useful as this will minimize optical diffraction andother optical interferences, and hence make the cell-culture substratematerial more transparent/translucent.

Shown schematically in FIG. 35 is a method for nano imprint patterningof a resist layer and etch-patterning the transparent glass or polymersubstrate through the resist. Once a liquid resist layer (such asspin-coated or dip-coated) is cured by heat or UV light while beingimpressed, the layer can be reactive ion etched (RIE) to remove anyresidual material near the bottom of the impressed cavity so that theplastic or glass substrate is exposed and can now be etched by RIE orchemical etching for substrate patterning. The pattern can be eitherprotruding or recessed, can be circular, oval, rectangular, or linearray pattern. The resist layer can be then removed and a thin, almosttransparent Ti or TiO₂ coating is deposited to obtain bio-compatible andcell-culture-enhancing transparent (or near transparent) substrates. Thenano pattern can be made periodic or random, depending on the need forminimizing light diffraction and interference.

Shown in FIG. 36 is an alternative method of fabricating transparent ortranslucent cell-culture substrate by nano or micro imprint based resistpattern transfer followed by chemical or RIE etching, plus coating of athin layer of Ti, TiO₂ or related metals or oxides. Nano or microimprint stamp picks up ink or paste containing chemical-etch-resistantresist material and transfers onto the surface of a plastic or glasssubstrate to be imprint. Similarly as in FIG. 35, the pattern can beperiodic or random, and can have either protruding or recessed, and canbe circular, oval, rectangular, or line array pattern. The substrate isthen chemical or RIE etched to form a pattern and a thin film of Ti orTiO₂ (or related metals, alloys, oxides, nitrides, etc.) is deposited toobtain bio-compatible and cell-culture-enhancing transparent (or neartransparent) substrates.

The nano or micro stamp for imprinting can be obtained by variouspatterned etch process, such as by electron beam lithography. FIG. 37 isan SEM micrograph showing an example silicon nano imprint stampcontaining 25 nm diameter periodic pillar array, which is suitable forimprinting process to fabricate a patterned cell-culture-substrate asdescribed in, e.g., FIGS. 33 to 36.

The invention provides biocompatible and cell-growth-enhancing culturesubstrate comprising elastically compliant protruding nanostructuresubstrate coated with Ti, TiO₂ or related metal and metal oxide films,as described herein. Exemplary nanostructure substrates of the inventioncan comprise a Ti, Zr, Hf, Nb, Ta, Mo and/or W metal material, a Ti, Zr,Hf, Nb, Ta, Mo and/or W alloy, a Ti, Zr, Hf, Nb, Ta, Mo and/or W oxideor nitride, and/or stainless steel or ceramic.

7. Biocompatible and Cell-Growth-Enhancing Culture Substrate ComprisingElastically Compliant Protruding Nanostructure Substrate Coated with Ti,TiO₂ or Related Metal and Metal Oxide Films

The invention provides biocompatible and cell-growth-enhancing culturesubstrate comprising elastically compliant nanostructures coated withTi, TiO₂ or related metal and metal oxide films, or in alternativeembodiments, a Ti, Zr, Hf, Nb, Ta, Mo and/or W metal material, a Ti, Zr,Hf, Nb, Ta, Mo and/or W alloy, a Ti, Zr, Hf, Nb, Ta, Mo and/or W oxideor nitride, and/or stainless steel or ceramic material. The coating canbe multi-laminar. The elastically compliant nanostructures of theinvention ameliorate the stress or strain that the growing/propagatingcells experience; the elastically compliant nanostructures have abeneficial effect on cell viability and growth behavior. By providing anelastically soft substrate, which is made even more flexible by virtueof protruding surface nanostructure, elastically compliantnanostructures of the invention can further enhance cell growth andviability.

The drawing shown as FIG. 38 represents a schematic illustration of anexample method for fabricating elastomer-based cell culture substratecured while imprinted using UV light or heat. The master pattern intowhich the elastomer is impressed into can be a recessed-cavity-patterntype so that the imprinted elastomer has protruding features; oralternatively, a recessed-pattern elastomer culture substrate is used.The pattern can be periodic or random, and can be circular, oval,rectangular, or line array pattern.

An exemplary method of making a biocompatible and cell-growth-enhancingculture substrate of this invention comprises an elastically compliantprotruding nanostructure substrate coated with, e.g., Ti, TiO₂ orrelated metal and metal oxide films, and the methods comprises providinga surface nano-patterned stamp, impressing into a wet, uncured elastomerlayer with the nanostamp, curing the polymer while being impressed bythermal curing or UV light curing, releasing and removing the stamp, anddepositing a thin film of, e.g., Ti, Zr, Hf, Nb, Ta, Mo or W metalmaterial, a Ti, Zr, Hf, Nb, Ta, Mo or W alloy, a Ti, Zr, Hf, Nb, Ta, MoW oxide, or Ti, Zr, Hf, Nb, Ta, Mo or W nitride, or equivalentcompounds, by a physical or chemical thin film deposition method.

In FIG. 38(a), the stamp is press imprinted to the wet, uncured polymerresist layer UV-curable or heat-curable elastomer, such as PDMS(polydimethylsiloxane). The PDMS layer is cured while being imprinted byUV light illumination through transparent substrate (from the bottom) orby heating (FIG. 38(b)). The stamp is then release to obtainnano-imprinted polymer pattern (FIG. 38(c)), and then is coated with Ti,TiO₂ or related bio-compatible surface coating (FIG. 38(d)). The cured,nanopatterned elastomer is then peeled off and used for cell culture(FIG. 38(e)), in vitro for cell culture supply or in-vivo as a part ofan implant structure.

Shown in FIG. 39 is an example SEM (scanning electron microscopy)structure of nano-imprinted, recessed cavity array pattern on PDMS(polydimethylsiloxane) obtained by nano imprinting using aprotruding-pillar-array metal stamp and UV light curing. FIG. 40represents an SEM micrograph showing a nanopatterned PDMS(polydimethylsiloxane) cell-culture substrate with protruding array ofsoft and compliant balls of about 200 nm diameter, which was obtained byusing a recessed-cavity-array silicon stamp and UV curing.

The various types of surface nanostructured or micro-structuredcell-culture or bone-culture substrates described in this invention canbe used either for in vitro culture of cells and bones, or as a part ofin vivo implant or drug or biological material delivery structure.

The types of cells or hard tissues that can be cultured in an enhancedmanner by the substrates of this invention include osteoblast cells,periodontal cells, hepatocyte (or mixed cells for liver cell culture),kidney cells, blood vessel cells, skin cells, stem cells, endothelialcells and other rare cells, as well as rapid formation/growth ofstrongly adherent bones. The structures according to the invention canbe useful for reliable and faster orthopedic or dental bone repair, forpreparation of partial or full implant organs for in-vivo insertion, orex-vivo operation as artificial lever or kidney, for externallycontrollable drug release and therapeutic treatments, for efficienttoxicity testing of drugs and chemicals, and for diagnosis/detection ofdisease or forensic cells.

Alternative embodiments of the invention include:

1. Biomaterials with Strongly Bonded, Protruding Features

The macroscopically or microscopically extended biomaterial topographyprovides a lock-in mechanical integrity at the implant-hard tissueinterface, while the TiO₂ nanotube type nano structure on the surface ofthe protrusion features and the surface of the base implant provides adesirable cell- or bone-growth-accelerating characteristics.

The invention provides various surface-protruding structures includinghairy wire or mesh screen Ti protrusion structure with surface nanoporeor nanotube TiO₂, particle- or fiber-aggregate protrusion structure,protrusion+implant base with re-entrant holes, fabrication techniques,method of bonding the protrusion structure to the base implant bydiffusion bonding, melt-bonding, spot welding, and applications formechanically locked-in, strongly adhered cell growth or bone growth,drug toxicity testing, artificial organs, dental or periodontalapplications.

2. Externally and remotely controllable drug-delivery systems—Inalternative embodiments, the nanotube or nanopore arrays, or micro-wiresor micro-ribbon arrays on the implant surface are utilized as areservoir for drug and other biological agents, with an advantageouscharacteristics of magnetically actuated, on-demand drug releasecapability. In alternative embodiments, the structures of the inventioncomprise various drug-reservoir nanopore structures+drugs/biologicalagents+optional magnetic nanoparticles. The invention provides remotecontrolled drug release mechanisms based on movement of magneticparticles versus high frequency (alternating current) AC magnetic fieldinduced hyperthermia heating effect. The nanostructures include particleaggregate, mesoporous structure, nanowire or ribbon forest,directionally etched porous materials, and porous thin films with theincorporated drugs/biological agents+magnetic nanoparticles. Variousfabrication/processing methods, and biomedical applications.

Elastically compliant implant material for bone growth—In alternativeembodiments, the invention provides subdivided, spring-like fiber ormesh screen shape implants for stress-accommodation and minimalseparation failures at the implant-hard tissue interface, which alsoprovides strength and toughness reinforcement of the grown bone viabone-metal wire composite formation. The invention provides elasticallycompliant implant structures, fabrication methods, strong-interface bonegrowth applications.

4. Non-metallic or non-Ti based substrates the surfaces of which havebeen converted to TiO₂ type nanotubes or nanopores—In alternativeembodiments, the invention provides products of manufacture comprising athin film coating of Ti or TiO₂, or thin, a macro or a microscalecoating comprising a Ti, Zr, Hf, Nb, Ta, Mo and/or W metal material, aTi, Zr, Hf, Nb, Ta, Mo and/or W alloy, a Ti, Zr, Hf, Nb, Ta, Mo and/or Woxide or nitride, and/or stainless steel or ceramic, applied onto thesurface(s) of already nanoporous material. In alternative embodiments,the term “thin” means having a thickness of at least about 1, 2, 3, 4,5, 6, 7, 8, 9 or 10 or more nm, or having a thickness of between about 1to 10 nm, or having a thickness of between about 1 to 15 nm, or having athickness of between about 1 to 20 nm. Alternatively, the inventionprovides products of manufacture comprising a thick film coating of Tior TiO₂, or a “thick” film comprising a macro or a micro scale coatingcomprising a Ti, Zr, Hf, Nb, Ta, Mo and/or W metal material, a Ti, Zr,Hf, Nb, Ta, Mo and/or W alloy, a Ti, Zr, Hf, Nb, Ta, Mo and/or W oxideor nitride, and/or stainless steel or ceramic, deposited and anodized tocreate a nanotube, e.g., a TiO₂ nanotube type, to exhibit a desirablecell or bone growth accelerating characteristics. The invention providesporous or patterned substrates which have been made biocompatible andcell- or bone-growth-accelerating by TiO₂ surface nanotubes, etc., andvarious fabrication methods, and biomedical applications.

5. Biocompatible materials configured in loose particles, looseshort-fibers, or loose flakes—The powder surfaces are processed tocomprise nanopore or nanotube array nanostructure, so that the loosepowders exhibit cell- or bone-growth-accelerating characteristics, whichis useful for bone cement and other tissue connection applications.Claims to be incorporated—Various types of fabrication methods for TiO₂surface nanotubes on loose powders, short-fibers, flakes, fragmentedmesh screens. Also, the invention provides various application methods,and biomedical applications including accelerated bone growth, dentalbone growth, periodontal tissue growth.

It should be understood that the invention can be practiced withmodification and alteration within the spirit and scope of the appendedclaims. The description is not intended to be exhaustive or to limit theinvention to the precise form disclosed. It should be understood thatthe invention can be practiced with modification and alteration and thatthe invention be limited only by the claims and the equivalents thereof.

1-58. (canceled)
 59. A product of manufacture, comprising: a substrate;and a layer, the layer comprising a biocompatible material with aplurality of nanotubes that have a diameter of at least 100 nm and havea height of at least 100 nm.
 60. The product of manufacture of claim 59,wherein the plurality of nanotubes are arranged on a surface of thelayer at a density of at least 0.2×10⁸/cm².
 61. The product ofmanufacture of claim 59, wherein the plurality of nanotubes arevertically aligned and laterally spaced.
 62. The product of manufactureof claim 59, wherein the substrate is a radiotranslucent thermoplasticpolymer and the layer is a thin film nanostructure.
 63. The product ofmanufacture of claim 59, wherein the biocompatible material is abiocompatible surface layer comprising any of a metal, metal alloy,titanium, titanium alloy, ceramic, titanium oxide, titanium dioxide,aluminum oxide, and any combination thereof.
 64. The product ofmanufacture of claim 59, wherein the substrate is a non-metallicsubstrate comprising any of a polymer, a thermosetting polymer, a porouspolymer, a plastic material, an elastomeric polymer, a transparentthermoplastic polymer, a transparent UV-light curable polymer, atransparent glass, a polydimethysiloxane (PDMS), a polymethylmethacrylate (PMMA), polyethene, polypropene, polystyrene, poly vinylchloride, equivalent compounds, and any combination thereof.
 65. Theproduct of manufacture of claim 59, wherein the layer is coupled to thesubstrate by applying the layer onto a surface of the substrate usingany of a thin-film and a thick-film coating followed by an anodizationprocess.
 66. The product of manufacture of claim 65, wherein theapplying further comprising depositing the layer onto the surface. 67.The product of manufacture of claim 65, wherein the anodization processcomprising an electrochemical anodization process followed by a heattreatment.
 68. The product of manufacture of claim 67, wherein the heattreatment crystallizes the plurality of nanotubes.
 69. The product ofmanufacture of claim 59, wherein the layer further comprises amicro-nano combined structure, the -nano component of the micro-nanocombined structure comprising the plurality of nanotubes as set forth inclaim
 59. 70. The product of manufacture of claim 59, wherein the layerfurther comprises a macro-nano combined structure, the -nano componentof the macro-nano combined structure comprising the plurality ofnanotubes as set forth in claim
 59. 71. The product of manufacture ofclaim 70, wherein the -macro component of the macro-nano combinedstructure comprises any of mesh screens, ribbons, and wire arrays. 72.The product of manufacture of claim 59, wherein the layer includes anyof growth factors, biological agents, antibiotics, genes, proteins,drugs, and magnetic nanoparticles associated with the plurality ofnanotubes.
 73. The product of manufacture of claim 59, wherein theplurality of nanotubes comprises any of Ti, Ti oxide, Zr, Hf, Nb, Ta,Mo, W, their alloys, their oxides, and any combination thereof.
 74. Theproduct of manufacture of claim 59, wherein the plurality of nanotubesare covered with TiO₂.
 75. The product of manufacture of claim 59,wherein the plurality of nanotubes comprises an elastically compliantnanostructure.
 76. An implant, comprising: a substrate; and a layer, thelayer comprising a biocompatible material with a plurality of nanotubesthat have a diameter of at least 100 nm and have a height of at least100 nm.
 77. The implant of claim 76, wherein the implant is any of adental implant, a spinal implant, and an orthopedic implant.
 78. Amethod for making a product of manufacture, comprising: providing asubstrate; and coupling a layer to the substrate, the layer comprising abiocompatible material with a plurality of nanotubes that have adiameter of at least 100 nm and have a height of at least 100 nm.